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Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin
Abstract
Mammalian mRNAs lose and acquire proteins throughout their life span while undergoing processing, transport, translation, and decay. How translation affects messenger RNA (mRNA)-protein interactions is largely unknown. The pioneer round of translation uses newly synthesized mRNA that is bound by cap-binding protein 80 (CBP80)-CBP20 (also known as the cap-binding complex [CBC]) at the cap, poly(A)-binding protein N1 (PABPN1) and PABPC1 at the poly(A) tail, and, provided biogenesis involves pre-mRNA splicing, exon junction complexes (EJCs) at exon-exon junctions. Subsequent rounds of translation engage mRNA that is bound by eukaryotic translation initiation factor 4E (eIF4E) at the cap and PABPC1 at the poly(A) tail, but that lacks detectable EJCs and PABPN1. Using the level of intracellular iron to regulate the translation of specific mRNAs, we show that translation promotes not only removal of EJC constituents, including the eIF4AIII anchor, but also replacement of PABPN1 by PABPC1. Remarkably, translation does not affect replacement of CBC by eIF4E. Instead, replacement of CBC by eIF4E is promoted by importin beta (IMPbeta): Inhibiting the binding of IMPbeta to the complex of CBC-IMPalpha at an mRNA cap using the IMPalpha IBB (IMPbeta-binding) domain or a RAN variant increases the amount of CBC-bound mRNA and decreases the amount of eIF4E-bound mRNA. Our studies uncover a previously unappreciated role for IMPbeta and a novel paradigm for how newly synthesized messenger ribonucleoproteins (mRNPs) are matured.
... For example, all newly synthesized mRNAs are bound by the nuclear Cap Binding Complex (CBC), the Exon Junction Complex (EJC) and the nuclear poly(A) binding protein (PABPN1) (4)(5)(6). By contrast, mRNAs licensed for translation are depleted of EJC proteins, and have exchanged CBC and PABPN1 at the cap and poly(A) tail for the eukaryotic translation initiation factor 4E (eIF4E) and the cytoplasmic poly(A) binding protein (PABPC), respectively (6)(7)(8). Termination codons located more than 25-35 nt upstream from an Exon Junction Complex (EJC) are strong NMD signals (9). Following PTC detection by a pioneering ribosome, the SMG1 phosphatidylinositol 3kinase-related kinase (SMG1) and the EJC protein UP-Frameshift Mutation 1 homolog (UPF1) interact with the translation termination release factor-1 and -3 (eRF1 and eRF3, respectively) to form the SURF complex (10,11). ...
... NMD efficiency may be linked to the composition of messenger ribonucleoprotein particles (mRNPs). The CBC is displaced by eIF4E in a translation-independent manner (8,15). This exchange mechanism is driven by interactions of CBP80 with the nuclear import receptor Importin b (IMPb) and also presumably by mass action due to the high cytoplasmic concentrations of eIF4E (8,16). ...
... The CBC is displaced by eIF4E in a translation-independent manner (8,15). This exchange mechanism is driven by interactions of CBP80 with the nuclear import receptor Importin b (IMPb) and also presumably by mass action due to the high cytoplasmic concentrations of eIF4E (8,16). These rearrangements have important consequences for NMD; for example the interaction of CBC with UPF1 promotes assembly of the SURF complex and is important for activating NMD (17). ...
RNA surveillance by the Nonsense Mediated Decay (NMD) pathway eliminates potentially deleterious transcripts containing Premature Termination Codons (PTCs). The transition from a pioneering round of translation to steady state translation is hypothesized to be a major checkpoint in this process. One hallmark of mRNAs licensed for translation is the exchange of 7-methylguanosine cap binding proteins. However, mRNAs undergoing steady state translation are also NMD substrates, raising mechanistic questions about the NMD checkpoint. To test the role of cap binding proteins in NMD, we modulated the protein composition of cytoplasmic messenger ribonucleoprotein particles (mRNPs) with the naturally occurring macrolide rapamycin. We demonstrate that despite well-documented attenuation of cap-dependent mRNA translation, rapamycin can augment NMD. Rapamycin-treatment significantly reduces the levels of endogenous and exogenous PTC-containing mRNA isoforms in a dose- and UPF1- dependent manner. PTC-containing transcripts exhibit a shorter half-life upon rapamacyin-treatment as compared to non-PTC isoforms. Rapamycin also causes depletion of PTC-containing mRNA isoforms from polyribosomes, suggesting that actively translating ribosomes can transition between low and high NMD states. Importantly, mRNPs show depletion of eIF4E and retention of the nuclear Cap Binding Complex (CBC) in rapamycin-treated cells. Our data demonstrate that rapamycin potentiates pioneer-like mRNP context thereby decreasing NMD evasion.
... This ensures that only mature mRNPs are exported. In the cytoplasm, early mRNPs usually engage a first round of translation, also referred to as the pioneer round of translation which remodels the mRNP once again (Figure 2) (Maquat et al., 2010b;Sato and Maquat, 2009). While the CBC, as part of the early mRNP, is required for the initiation of the pioneer round of translation, translation in the steady state mode requires the eukaryotic initiation factor 4E (eIF4E). ...
... The replacement of the CBC by eIF4E occurs in a translation-independent manner and is mediated by Importin-α, which associates with the cap-bound CBC. Subsequently, its binding to Importin-β induces the dissociation of the CBC from the 5' cap which is then occupied by eIF4E (Sato and Maquat, 2009). The first translating ribosome displaces the EJC from the open reading frame (ORF) of the mRNP via interacting with the EJC removal factor PYM (Gehring et al., 2009). ...
... This exchange also depends on the translation. Therefore, PABPN1, CBC EJC and possibly many more factors are only present in early mRNPs, whereas eIF4E is part of mRNPs that have already completed the pioneer round of translation (Hosoda et al., 2006;Sato and Maquat, 2009). ...
The eukaryotic gene expression requires extensive regulations to enable the homeostasis of the cell and to allow dynamic responses due to external stimuli. Although many regulatory mechanisms involve the transcription as the first step of the gene expression, intensive regulation occurs also in the post-transcriptional mRNA metabolism. Thereby, the particular composition of the mRNPs plays a central role as the components associated with the mRNA form a specific “mRNP code” which determines the fate of the mRNA. Many proteins which are involved in this regulation and the mRNA metabolism are affected in diseases and especially neurological disorders often result from an aberrant mRNP code which leads to changes in the regulation and expression of mRNPs. The focus of this work was on a trimeric protein complex which is termed TTF complex based on its subunits TDRD3, TOP3β and FMRP. Biochemical investigations revealed that the three components of the TTF complex are nucleo-cytosolic shuttle proteins which localize in the cytoplasm at the steady-state, associate with mRNPs and are presumably connected to the translation. Upon cellular stress conditions, the TTF components concentrate in stress granules. Thus, the TTF complex is part of the mRNP code, however its target RNAs and function are still completely unknown. Since the loss of functional FMRP results in the fragile X syndrome and TOP3β is associated with schizophrenia and intellectual disability, the TTF complex connects these phenotypically related neuro-psychiatric disorders with each other on a molecular level. Therefore, the aim of this work was to biochemically characterize the TTF complex and to define its function in the mRNA metabolism. In this work, evidence was provided that TDRD3 acts as the central unit of the TTF complex and directly binds to FMRP as well as to TOP3β. Thereby, the interaction of TDRD3 and TOP3β is very stable, whereas FMRP is a dynamic component. Interestingly, the TTF complex is not bound directly to mRNA, but is recruited via the exon junction complex (EJC) to mRNPs. This interaction is mediated by a specific binding motif of TDRD3, the EBM. Upon biochemical and biological investigations, it was possible to identify the interactome of the TTF complex and to define the role in the mRNA metabolism. The data revealed that the TTF complex is mainly associated with “early” mRNPs and is probably involved in the pioneer round of translation. Furthermore, TOP3β was found to bind directly to the ribosome and thus, establishes a connection between the EJC and the translation machinery. A reduction of the TTF components resulted in selective changes in the proteome in cultured cells, whereby individual protein subsets seem to be regulated rather than the global protein expression. Moreover, the enzymatic analysis of TOP3β indicated that TOP3β is a type IA topoisomerase which can catalytically attack not only DNA but also RNA. This aspect is particularly interesting with regard to the connection between early mRNPs and the translation which has been revealed in this work. The data obtained in this work suggest that the TTF complex plays a role in regulating the metabolism of an early mRNP subset possibly in the course of the pioneer round of translation. Until now, the link between an RNA topoisomerase and the mRNA metabolism is thereby unique and thus provides a completely new perspective on the steps in the post-transcriptional gene expression and its regulation.
... Linear [6] 500 nm (250-800 nm range) [12] 100-300 nm [13] 300-1000 nm [14] a 90,000-150,000 [12] a 50,000-300,000 [15] YBX1 [11,12] PABPC1 [11] PABPN1 [57] hnRNPs [11] SR proteins [11] CBP20/CBP80 [57] EJCs [57] YES Translating mRNPs Linear [10,70] Circular [66,67] MDN1 mRNA (18 413 nt) = 135 nm POLA1 mRNA (5486 nt) = 96 nm PRPF8 mRNA (7295 nt) = 92 nm [10] a eIF4E [100] PABPC1 [100] Polyribosomes YES P-bodies Spherical [17] 150-240 nm [16] ∼500 nm [17] ∼4-7 (unstressed conditions) [17] LSM14A [17] DDX6 [17] 4E-T [17] DCP1 [17] EDC3 [17] No ribosomal subunits YES Increased number by stress agents or heat shock [22] Stress granules Irregular sphere Dynamic shell-like structure surrounding a stable core [27] 0.1-2 μm [25] 30-50 [24] (variable, depending on duration of stress) ...
... Linear [6] 500 nm (250-800 nm range) [12] 100-300 nm [13] 300-1000 nm [14] a 90,000-150,000 [12] a 50,000-300,000 [15] YBX1 [11,12] PABPC1 [11] PABPN1 [57] hnRNPs [11] SR proteins [11] CBP20/CBP80 [57] EJCs [57] YES Translating mRNPs Linear [10,70] Circular [66,67] MDN1 mRNA (18 413 nt) = 135 nm POLA1 mRNA (5486 nt) = 96 nm PRPF8 mRNA (7295 nt) = 92 nm [10] a eIF4E [100] PABPC1 [100] Polyribosomes YES P-bodies Spherical [17] 150-240 nm [16] ∼500 nm [17] ∼4-7 (unstressed conditions) [17] LSM14A [17] DDX6 [17] 4E-T [17] DCP1 [17] EDC3 [17] No ribosomal subunits YES Increased number by stress agents or heat shock [22] Stress granules Irregular sphere Dynamic shell-like structure surrounding a stable core [27] 0.1-2 μm [25] 30-50 [24] (variable, depending on duration of stress) ...
... Linear [6] 500 nm (250-800 nm range) [12] 100-300 nm [13] 300-1000 nm [14] a 90,000-150,000 [12] a 50,000-300,000 [15] YBX1 [11,12] PABPC1 [11] PABPN1 [57] hnRNPs [11] SR proteins [11] CBP20/CBP80 [57] EJCs [57] YES Translating mRNPs Linear [10,70] Circular [66,67] MDN1 mRNA (18 413 nt) = 135 nm POLA1 mRNA (5486 nt) = 96 nm PRPF8 mRNA (7295 nt) = 92 nm [10] a eIF4E [100] PABPC1 [100] Polyribosomes YES P-bodies Spherical [17] 150-240 nm [16] ∼500 nm [17] ∼4-7 (unstressed conditions) [17] LSM14A [17] DDX6 [17] 4E-T [17] DCP1 [17] EDC3 [17] No ribosomal subunits YES Increased number by stress agents or heat shock [22] Stress granules Irregular sphere Dynamic shell-like structure surrounding a stable core [27] 0.1-2 μm [25] 30-50 [24] (variable, depending on duration of stress) ...
Cytoplasmic messenger ribonucleoprotein particles (mRNPs) represent the cellular transcriptome, and recent data have challenged our current understanding of their architecture, transport, and complexity before translation. Pre‐translational mRNPs are composed of a single transcript, whereas P‐bodies and stress granules are condensates. Both pre‐translational mRNPs and actively translating mRNPs seem to adopt a linear rather than a closed‐loop configuration. Moreover, assembly of pre‐translational mRNPs in physical RNA regulons is an unlikely event, and co‐regulated translation may occur locally following extracellular cues. We envisage a stochastic mRNP transport mechanism where translational repression of single mRNPs—in combination with microtubule‐mediated cytoplasmic streaming and docking events—are prerequisites for local translation, rather than direct transport.
... All newly synthesized and spliced mRNAs are bound by the nuclear Cap Binding Complex (CBC), the Exon Junction Complex (EJC) and the nuclear poly(A) binding protein (PABPN1) (5)(6)(7). By contrast, mRNAs engaged in steady-state translation are depleted of EJC proteins, and have exchanged CBC and PABPN1 at the cap and poly(A) tail for the eukaryotic translation initiation factor 4E (eIF4E) and the cytoplasmic poly(A) binding protein (PABPC), respectively (7)(8)(9). Termination codons located more than 50-55 nt upstream from an Exon Junction Complex (EJC) are strong NMD signals (10). During mRNA processing, the SURF complex (SMG1, UPF1, eRF1 and eRF3) associates with the downstream EJC and triggers SMG1-dependent phosphorylation of UPF1, which is required for inhibition of translation initiation and subsequent nucleolytic decay of the targeted mRNA (11)(12)(13). ...
... NMD efficiency may be linked to the composition of messenger ribonucleoprotein particles (mRNPs), from those ac-quired during splicing to the cap-binding factors. The CBC is displaced by eIF4E in a translation-independent manner (9,16). This exchange mechanism is driven by interactions of CBP80 with the nuclear import receptor Importin  (IMPb) (9). ...
... The CBC is displaced by eIF4E in a translation-independent manner (9,16). This exchange mechanism is driven by interactions of CBP80 with the nuclear import receptor Importin  (IMPb) (9). It is possible that this exchange may also be regulated by mass action due to the high cytoplasmic concentrations of eIF4E (17). ...
Rapamycin is a naturally occurring macrolide whose target is at the core of nutrient and stress regulation in a wide range of species. Despite well-established roles as an inhibitor of cap-dependent mRNA translation, relatively little is known about its effects on other modes of RNA processing. Here, we characterize the landscape of rapamycin-induced post-transcriptional gene regulation. Transcriptome analysis of rapamycin-treated cells reveals genome-wide changes in alternative mRNA splicing and pronounced changes in NMD-sensitive isoforms. We demonstrate that despite well-documented attenuation of cap-dependent mRNA translation, rapamycin can augment NMD of certain transcripts. Rapamycin-treatment significantly reduces the levels of both endogenous and exogenous Premature Termination Codon (PTC)-containing mRNA isoforms and its effects are dose-, UPF1- and 4EBP-dependent. The PTC-containing SRSF6 transcript exhibits a shorter half-life upon rapamycin-treatment as compared to the non-PTC isoform. Rapamycin-treatment also causes depletion of PTC-containing mRNA isoforms from polyribosomes, underscoring the functional relationship between translation and NMD. Enhanced NMD activity also correlates with an enrichment of the nuclear Cap Binding Complex (CBC) in rapamycin-treated cells. Our data demonstrate that rapamycin modulates global RNA homeostasis by NMD.
... For instance, an aberrant mRNA harboring a premature termination codon (PTC) is rapidly degraded by nonsense-mediated mRNA decay (NMD), the bestcharacterized mRNA surveillance mechanism (23,24), with most of the degradation occurring during pioneer translation (22). In contrast, when an mRNA is normal and thus passes the mRNA surveillance pathway during pioneer translation, a cytoplasmic cap-binding protein called eukaryotic translation initiation factor 4E (eIF4E) replaces the CBC with the help of importins (IMPs) ␣ and  in a translation-independent manner (25)(26)(27). The resulting eIF4E-associated messenger ribonucleoprotein (mRNP) then participates in active protein synthesis. ...
... How does the inefficient binding of RNC-SRP to the SR lead to accumulation of the CBC-RNC-SRP complex? We noted that fewer CBP80 and IMP␣ were enriched in the IP of IMP following SR downregulation (Supplementary Figure S7A and B), implying that the CBC-RNC-SRP complex may not be favorable for IMP-mediated mRNP remodeling (25,26). ...
The pioneer (or first) round of translation of newly synthesized mRNAs is largely mediated by a nuclear cap-binding complex (CBC). In a transcriptome-wide analysis of polysome-associated and CBC-bound transcripts, we identify RN7SL1, a noncoding RNA component of a signal recognition particle (SRP), as an interaction partner of the CBC. The direct CBC–SRP interaction safeguards against abnormal expression of polypeptides from a ribosome–nascent chain complex (RNC)–SRP complex until the latter is properly delivered to the endoplasmic reticulum. Failure of this surveillance causes abnormal expression of misfolded proteins at inappropriate intracellular locations, leading to a cytosolic stress response. This surveillance pathway also blocks protein synthesis through RNC–SRP misassembled on an mRNA encoding a mitochondrial protein. Thus, our results reveal a surveillance pathway in which pioneer translation ensures proper targeting of endoplasmic reticulum and mitochondrial proteins.
... De plus, chez la levure, les queues des ARNm, à l'origine d'une taille d'environ 200 nucléotides, sont raccourcies jusqu'à 50-70 nucléotides par le complexe PAN2/PAN3, dont le fonctionnement dépend de PABPC1. La réalisation de ce processus est nécessaire à l'export des ARNm (Brown and Sachs 1998;Dunn et al. 2005 (Sato and Maquat 2009). Après cela, le complexe CBC est remplacé par eIF4E au niveau de la coiffe de l'ARNm, permettant l'initiation de la traduction proprement dite. ...
... Elle joue un rôle important en particulier dans la polyadénylation des ARNm au noyau et leur export dans le cytoplasme(Kühn et al. 2009). Elle reste liée à la queue polyA des ARNm jusqu'au début de leur traduction(Sato and Maquat 2009). Il est donc possible qu'elle interagisse avec M2-1 après sa sortie du noyau. ...
Bien que le Virus Respiratoire Syncytial, responsable de la bronchiolite du nourrisson, soit aujourd’hui un problème de santé publique majeur, il n’existe encore aucun vaccin ou antiviral curatif contre ce pathogène. Le manque de données sur les étapes clés du cycle viral et sur les interactions virus-cellule freine le développement de nouvelles molécules antivirales.Nous avons étudié l’interactome de deux protéines virales : la polymérase L et le facteur de transcription M2-1. Dans ce but, nous avons mis au point un crible s’appuyant à la fois sur des critères d’interactomique et sur des critères fonctionnels.La première étape consistait à identifier des partenaires potentiels de M2-1 et L par des co-immunoprécipitations couplées à une approche de protéomique quantitative. Pour plus de pertinence, ce crible a été réalisé sur cellules infectées, grâce des virus recombinants produits par génétique inverse. Ceci nous a permis d’identifier 45 et 137 partenaires potentiels de L et M2-1 respectivement. Une étude systématique de l’impact de l’inhibition de 15 partenaires potentiels de M2-1 sur la multiplication virale a mis en avant trois candidats : ILF2, PABPN1 et PABPC1.Nous nous sommes par la suite concentrés sur PABPC1. L’inhibition de l’expression de PABPC1 altère la multiplication virale, mais nous n’avons pas pu mettre en évidence un effet spécifique sur la transcription ou la traduction virale. Son interaction avec M2-1 a été confirmée, et le domaine MLLE de PABPC1 a été identifié comme le site de liaison à M2-1. L’interaction entre M2-1 et PABPC1 a été observée à la fois dans le cytoplasme et dans les IBAGs, des sous-structures concentrant les ARNm viraux au sein des corps d’inclusion viraux. Nous avons formulé l’hypothèse que M2-1, liée à PABPC1, accompagne les ARNm viraux après leur sortie des corps d’inclusion. Ceci suggère un rôle de M2-1 dans le devenir des ARNm viraux en aval de leur transcription.
... Replacement indices were determined by normalizing the relative levels of co-IPed reporter mRNAs in the IP of eIF4E to those in the IP of CBP80. histone mRNAs and U1 small nuclear RNA (snRNA) in the IP of cytoplasmic CBP80 (42,43), a greater amount of those mRNAs turned out to be enriched in the IP of CBP80 relative to the IP of eIF4E ( Figure 1C and D). In the same IPs, comparable amounts of all the tested reporter mRNAs were found to be enriched in the IPs of either CBP80 or eIF4E ( Figure 1E). ...
... Then, the IMP␣-CBC complex binds to the cap of newly synthesized RNAs, again forming the IMP␣-CBC-capped mRNA complex. Of note, it is known that the IMP-mediated replacement of the CBC by eIF4E occurs in a translation-independent manner (43). ...
Newly synthesized mRNAs are exported from the nucleus to cytoplasm with a 5'-cap structure bound by the nuclear cap-binding complex (CBC). During or after export, the CBC should be properly replaced by cytoplasmic cap-binding protein eIF4E for efficient protein synthesis. Nonetheless, little is known about how the replacement takes place. Here, we show that double-stranded RNA-binding protein staufen1 (STAU1) promotes efficient replacement by facilitating an association between the CBC-importin α complex and importin β. Our transcriptome-wide analyses and artificial tethering experiments also reveal that the replacement occurs more efficiently when an mRNA associates with STAU1. This event is inhibited by a key nonsense-mediated mRNA decay factor, UPF1, which directly interacts with STAU1. Furthermore, we find that cellular apoptosis that is induced by ionizing radiation is accompanied by inhibition of the replacement via increased association between STAU1 and hyperphosphorylated UPF1. Altogether, our data highlight the functional importance of STAU1 and UPF1 in the course of the replacement of the CBC by eIF4E, adding a previously unappreciated layer of post-transcriptional gene regulation.
... Translation initiation is a rate-limiting and multi-step process involving a large number of initiation factors (Figure 1a). During transcription, the nuclear capbinding complex (CBC), consisting of CBP80 and CBP20, binds the cap structure (m 7 GpppN) of precursor mRNAs and subsequently escorts the mature mRNAs from the nucleoplasm to the cytoplasm [1]. CBC-bound mRNA undergoes a pioneer round of translation, in which premature stop codon-containing transcripts can be identified and degraded by the nonsensemediated mRNA decay (NMD) surveillance pathway [2]. ...
... CBC-bound mRNA undergoes a pioneer round of translation, in which premature stop codon-containing transcripts can be identified and degraded by the nonsensemediated mRNA decay (NMD) surveillance pathway [2]. Subsequently, the exchange of the CBC for eIF4E takes place through the binding of the nuclear transport receptor importin-β to the CBC-importin-α complex [1]. The cap-bound eIF4E, in conjunction with the RNA helicase eIF4A and scaffold protein eIF4G, forms the eIF4F complex. ...
Protein synthesis is tightly regulated, and its dysregulation can contribute to the pathology of various diseases, including cancer. Increased or selective translation of mRNAs can promote cancer cell proliferation, metastasis and tumor expansion. Translational control is one of the most important means for cells to quickly adapt to environmental stresses. Adaptive translation involves various alternative mechanisms of translation initiation. Upstream open reading frames (uORFs) serve as a major regulator of stress-responsive translational control. Since recent advances in omics technologies including ribo-seq have expanded our knowledge of translation, we discuss emerging mechanisms for uORF-mediated translation regulation and its impact on cancer cell biology. A better understanding of dysregulated translational control of uORFs in cancer would facilitate the development of new strategies for cancer therapy.
... In addition to the classical roles of importin-β/α in nucleocytoplasmic transport, importin-β and importin-α exert important roles in cap-binding activity. Importin-α partakes in CBC in the nucleus and importin-β triggers the replacement of CBCimportin-α complex by eIF-4E at mRNA caps when they exit the nuclear pore [213][214][215]. Ranbp1 and/or Ranbp2, and possibly with the cooperation of RanGAP, triggers the liberation of importin-β from Ran-GTP after exiting the nuclear pore and thus allows importin-β to engage with the capped mRNP-CBC-importin-α complex in the cytosolic compartment. ...
... First, CBC-capped mRNPs undergo a pioneer round of translation, which serves as a quality control mechanism to survey mRNAs destined for premature termination of translation and/or nonsense-mediated mRNA decay [216][217][218][219]. This step also promotes the remodeling of mRNPs by uncoating spliced mRNAs from other components, such as exon junction complex proteins [214,220]. Second, the replacement of the CBC-capped mRNA by eIF-4E promotes the steady-state initiation of translation and protein synthesis. Finally, CBC is strongly regulated by growth factors and environmental stressors that by this mechanism regulate gene expression (e.g., suppression of translation) [215,[221][222][223]. ...
The nuclear pore is the gatekeeper of nucleocytoplasmic transport and signaling through which a vast flux of information is continuously exchanged between the nuclear and cytoplasmic compartments to maintain cellular homeostasis. A unifying and organizing principle has recently emerged that cements the notion that several forms of amyotrophic lateral sclerosis (ALS), and growing number of other neurodegenerative diseases, co-opt the dysregulation of nucleocytoplasmic transport and that this impairment is a pathogenic driver of neurodegeneration. The understanding of shared pathomechanisms that underpin neurodegenerative diseases with impairments in nucleocytoplasmic transport and how these interface with current concepts of nucleocytoplasmic transport is bound to illuminate this fundamental biological process in a yet more physiological context. Here, I summarize unresolved questions and evidence and extend basic and critical concepts and challenges of nucleocytoplasmic transport and its role in the pathogenesis of neurodegenerative diseases, such as ALS. These principles will help to appreciate the roles of nucleocytoplasmic transport in the pathogenesis of ALS and other neurodegenerative diseases, and generate a framework for new ideas of the susceptibility of motoneurons, and possibly other neurons, to degeneration by dysregulation of nucleocytoplasmic transport.
... This complex is very important for the subsequent recruitment of other complexes, such as the spliceosome (32,33), the transcription/export (TREX) complex (34), or the RNA decay machinery (35). In addition, after maturation of the mRNA, CBC translocates to the cytoplasm in association with the mRNP and localizes to the polysomes, where it supports the first round of translation (36)(37)(38). During this step, there is an extensive remodeling of mRNP's composition, starting with the replacement of CBC by the eIF4E cap-binding protein (38,39). ...
... In addition, after maturation of the mRNA, CBC translocates to the cytoplasm in association with the mRNP and localizes to the polysomes, where it supports the first round of translation (36)(37)(38). During this step, there is an extensive remodeling of mRNP's composition, starting with the replacement of CBC by the eIF4E cap-binding protein (38,39). ...
Epstein-Barr virus (EBV) expresses several mRNAs produced from intronless genes that could potentially be unfavorably translated compared to cellular spliced mRNAs. To overcome this situation, the virus encodes an RNA-binding protein (RBP) called EB2, which was previously found to both facilitate the export of nuclear mRNAs and increase their translational yield. Here, we show that EB2 binds both nuclear and cytoplasmic cap-binding complexes (CBC and eukaryotic initiation factor 4F [eIF4F], respectively) as well as the poly(A)-binding protein (PABP) to enhance translation initiation of a given messenger ribonucleoparticle (mRNP). Interestingly, such an effect can be obtained only if EB2 is initially bound to the native mRNPs in the nucleus. We also demonstrate that the EB2-eIF4F-PABP association renders translation of these mRNPs less sensitive to translation initiation inhibitors. Taken together, our data suggest that EB2 binds and stabilizes cap-binding complexes in order to increase mRNP translation and furthermore demonstrate the importance of the mRNP assembly process in the nucleus to promote protein synthesis in the cytoplasm.
... This complex is very important for the subsequent recruitment of other complexes, such as the spliceosome (32,33), the transcription/export (TREX) complex (34), or the RNA decay machinery (35). In addition, after maturation of the mRNA, CBC translocates to the cytoplasm in association with the mRNP and localizes to the polysomes, where it supports the first round of translation (36)(37)(38). During this step, there is an extensive remodeling of mRNP's composition, starting with the replacement of CBC by the eIF4E cap-binding protein (38,39). ...
... In addition, after maturation of the mRNA, CBC translocates to the cytoplasm in association with the mRNP and localizes to the polysomes, where it supports the first round of translation (36)(37)(38). During this step, there is an extensive remodeling of mRNP's composition, starting with the replacement of CBC by the eIF4E cap-binding protein (38,39). ...
Epstein-Barr virus (EBV) expresses several mRNAs produced from intronless genes that could potentially be unfavorably translated compared to cellular spliced mRNAs. To overcome this situation, the virus encodes an RNA-binding protein (RBP) called EB2, previously found to both facilitate the export of nuclear mRNAs and increase their translational yield. Here, we show that EB2 binds both nuclear and cytoplasmic cap-binding complexes (respectively, CBC and eIF4F) as well as the poly(A)-binding protein (PABP) to enhance translation initiation of a given mRNP. Interestingly, such an effect can only be obtained if EB2 is initially bound to the native mRNPs in the nucleus. We also demonstrate that the EB2-eIF4F-PABP association renders translation of these mRNPs less sensitive to translation initiation inhibitors. Taken together, our data suggest that EB2 binds and stabilizes cap-binding complexes in order to increase mRNP translation and furthermore demonstrates the importance of the mRNP assembly process in the nucleus to promote protein synthesis in the cytoplasm.
IMPORTANCE Most herpesvirus early and late genes are devoid of introns. However, it is now well documented that mRNA splicing facilitates recruitment on the mRNA of cellular factors involved in nuclear mRNA export and translation efficiency. To overcome the absence of splicing of herpesvirus mRNAs, a viral protein - EB2 in the case of Epstein-Barr virus - is produced to facilitate the cytoplasmic accumulation of viral mRNAs. Although we previously showed that EB2 also specifically enhances translation of its target mRNAs, the mechanism was unknown. Here, we show that EB2 is first recruited to the mRNA cap structure in the nucleus, then interacts with the eIF4G and PABP proteins to enhance the initiation step of translation.
... Efficient NMD requires at least a single round of translation to recognize the PTCs on mRNAs. NMD has long been considered to occur during CT because (i) CBC-bound mRNAs are precursors of eIF4E-bound mRNAs, (ii) the CBC can drive cap-dependent translation, and (iii) the majority of NMD occurs on CBC-bound mRNAs (7,8,20,49,54,55). Therefore, the traditional model of CT-coupled NMD can be described as follows: newly synthesized CBC-bound and EJC-deposited mRNAs are subject to the first round of translation during or after their export from the nucleus to the cytoplasm. ...
... However, another equally plausible explanation is that CBC-bound NMD substrates may be predominantly subject to EJC-dependent NMD, whereas eIF4Ebound NMD substrates may be subject to EJC-independent NMD. Fourth, the replacement of the CBC by eIF4E occurs in a translation-independent manner (55). Therefore, even before the complete termination of CT, CBC would be replaced by eIF4E, suggesting the possibility of a transient interaction between eIF4E and UPF1. ...
In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: CBC-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap-dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance, whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in translational regulation. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation.
... In addition, the incorporation or not of the extra CAG sequence at the transcript level can affect the strength of some interactions. Of note, these interaction partners have known functions in RNA biology: UPF1 is best known for its central role in NMD among other functions (2), STRAP is implicated in different aspects of RNA metabolism (51)(52)(53), PABPC1 interacts with the polyA tail of mRNAs (62), and C1QBP is a multifunctional protein with a role in RNA splicing and homologous recombination (63)(64)(65)(66). ...
Many transcripts are targeted by nonsense-mediated decay (NMD), leading to their degradation and the inhibition of their translation. We found that the protein SUZ domain-containing protein 1 (SZRD1) interacts with the key NMD factor up-frameshift 1 (UPF1). When recruited to NMD-sensitive reporter gene transcripts, SZRD1 increased protein production, at least in part, by relieving translational inhibition. The conserved SUZ domain in SZRD1 was required for this effect. The SUZ domain is present in only three other human proteins besides SZRD1 : R3H-domain-containing protein 1 and 2 (R3HDM1, R3HDM2) and cAMP-regulated phosphoprotein 21 (ARPP21). We found that ARPP21, similarly to SZRD1, can increase protein production from NMD-sensitive reporter transcripts in a SUZ domain-dependent manner. This indicated that the SUZ domain-containing proteins could prevent translational inhibition of transcripts targeted by NMD. Consistent with the idea that SZRD1 mainly prevents translational inhibition, we did not observe a systematic decrease in the abundance of NMD targets when we knocked down SZRD1. Surprisingly, knockdown of SZRD1 in two different cell lines led to reduced levels of the NMD component UPF3B, which was accompanied by increased levels in a subset of NMD targets. This suggests that SZRD1 is required to maintain normal UPF3B levels and indicates that the effect of SZRD1 on NMD targets is not limited to a relief from translational inhibition. Overall, our study reveals that human SUZ-domain-containing proteins play a complex role in regulating protein output from transcripts targeted by NMD.
... 2005). The CBC-eIF4E handover has been reported to be catalysed by components of the nuclear export machinery (Sato and Maquat, 2009). Moreover, there is an emerging role for RGG-motif containing proteins, which also begin to interact with mRNPs around the time of mRNA export (Poornima et al., 2021), in both CBC-eIF4E and eIF4E-decapping complex handover events (Chowdhury and Jin, 2022). ...
In vitro transcribed, modified messenger RNAs (IVTmRNAs) have been used to vaccinate billions of individuals against the SARS-CoV-2 virus, and are currently being developed for many additional therapeutic applications. IVTmRNAs must be translated into proteins with therapeutic activity by the same cellular machinery that also translates native endogenous transcripts. However, different genesis pathways and routes of entry into target cells as well as the presence of modified nucleotides mean that the way in which IVTmRNAs engage with the translational machinery, and the efficiency with which they are being translated, differs from native mRNAs. This review summarises our current knowledge of commonalities and differences in translation between IVTmRNAs and cellular mRNAs, which is key for the development of future design strategies that can generate IVTmRNAs with improved activity in therapeutic applications.
... The early/pioneer rounds of translation occurring on newly exported mRNAs, as part of mRNA quality control processes such as nonsense codon scanning, involve an extensive remodeling of RNP composition, where nuclear cap and polyA tail binding proteins are exchanged, exonjunction complex proteins displaced as are numerous prion/aggregation domain-bearing nuclear RNA binding proteins, which are then imported back into the nucleus by the nuclear import machinery [84][85][86][87][88]. Under stress conditions where translation initiation is inhibited, newly exported mRNAs would be compromised in their ability to undergo such RBP remodeling. ...
Stress granules (SGs), structurally dynamic, optically resolvable, macromolecular assemblies of mRNAs, RNA binding proteins (RBPs), translation factors, ribosomal subunits, as well as other interacting proteins, assemble in response to cell stress conditions that elicit phosphorylation of eukaryotic initiation factor 2α (eIF2α) and consequently, the inactivation of translation initiation. SG biology is conserved throughout eukaryotes and has recently been linked to the pathological sequelae of neurodegenerative disorders, cancer biology, and viral infection. Substantial insights into mechanisms of SG biogenesis, and more broadly the phenomenon of biological liquid-liquid phase separation (LLPS), have been aided by detailed proteomic and transcriptomic studies as well as in vitro reconstitution approaches. A particularly interesting and largely unexplored element of SG biology is the cell biological context of SG biogenesis, including its subcellular organization and more recently, evidence that the endoplasmic reticulum (ER) membrane may serve important functions in RNA granule biology generally and SG biogenesis specifically. A central role for the ER in SG biogenesis is discussed and a hypothesis linking SG formation on the ER to the trafficking, localization and de novo translation of newly exported mRNAs is presented.
... Replacing PABPNs with PABPCs can occur after mRNAs are transported into the cytoplasm, especially during the first round of mRNA translation, which appears to promote the process. Thus, PABPN and PABPC conversion signals might be related to the translation efficiency of a transcript [30,31]. ...
The alga Chlamydomonas reinhardtii is a potential platform for recombinant protein expression in the future due to various advantages. Dozens of C. reinhardtii strains producing genetically engineered recombinant therapeutic protein have been reported. However, owing to extremely low protein expression efficiency, none have been applied for industrial purposes. Improving protein expression efficiency at the molecular level is, therefore, a priority. The 3′-end poly(A) tail of mRNAs is strongly correlated with mRNA transcription and protein translation efficiency. In this study, we identified a canonical C. reinhardtii poly(A) polymerase (CrePAPS), verified its polyadenylate activity, generated a series of overexpressing transformants, and performed proteomic analysis. Proteomic results demonstrated that overexpressing CrePAPS promoted ribosomal assembly and enhanced protein accumulation. The accelerated translation was further verified by increased crude and dissolved protein content detected by Kjeldahl and bicinchoninic acid (BCA) assay approaches. The findings provide a novel direction in which to exploit photosynthetic green algae as a recombinant protein expression platform.
... The transition from PABPN to PABPC on the tail is not well understood. Reporter studies have suggested that translation may facilitate this transition (20). PABPC supports numerous protein interactions that promote translation and stability such as binding to the translation initiation factor eIF4G and the translation termination factor eRF3 (21)(22)(23)(24)(25)(26)(27)(28). ...
The poly(A)-tail appended to the 3′-end of most eukaryotic transcripts plays a key role in their stability, nuclear transport, and translation. These roles are largely mediated by Poly(A) Binding Proteins (PABPs) that coat poly(A)-tails and interact with various proteins involved in the biogenesis and function of RNA. While it is well-established that the nuclear PABP (PABPN) binds newly synthesized poly(A)-tails and is replaced by the cytoplasmic PABP (PABPC) on transcripts exported to the cytoplasm, the distribution of transcripts for different genes or isoforms of the same gene on these PABPs has not been investigated on a genome-wide scale. Here, we analyzed the identity, splicing status, poly(A)-tail size, and translation status of RNAs co-immunoprecipitated with endogenous PABPN or PABPC in human cells. At steady state, many protein-coding and non-coding RNAs exhibit strong bias for association with PABPN or PABPC. While PABPN-enriched transcripts more often were incompletely spliced and harbored longer poly(A)-tails and PABPC-enriched RNAs had longer half-lives and higher translation efficiency, there are curious outliers. Overall, our study reveals the landscape of RNAs bound by PABPN and PABPC, providing new details that support and advance the current understanding of the roles these proteins play in poly(A)-tail synthesis, maintenance, and function.
... MARVELD1 is a microtubule-associated protein that can significantly inhibit cell proliferation, promote G1 phase cell-arrest, and reduce cell migration in mice [30]. Moreover, MARVELD1 plays a role in the pre-mRNA processing of integrin β1, thereby regulating cell adhesion and cell movement, a process paralleled to nonsense-mediated mRNA decay (NMD) and associated with the potential NMD factor Importin b1 [31][32][33]. Reducing MARVELD1 levels in lung cancer tissues also reduces the efficiency of NMD by diminishing the link between the UPF1/SMG1 components of the NMD complex and the mRNA containing a premature stop codon (PTC-mRNA) [18,34]. These reports suggest that the function of MARVELD1 could be related to the NMD regulatory signaling pathway. ...
MARVEL domain-containing 1 (MARVELD1) is one of the MARVEL domain-containing proteins. Expression of MARVELD1 in tumor and non-tumor tissues, the relationship between its expression and cancer prognosis, and upstream regulation of MARVELD1 were examined using pan-cancer data from The Cancer Genome Atlas. MARVELD1 expression was significantly downregulated in tissues used for pan-cancer analysis compared to that in normal tissues. Low expression of MARVELD1 was associated with poor disease outcomes in pan-cancer. Colon cancer patients with low expression of MARVELD1 had worse progression free survival and overall survival than those with high expression levels in our cohort. Hypermethylation and histone modification in the MARVELD1 promoter locus synergistically affected its expression in pan-cancer. The function of MARVELD1 in colon cancer remains to be studied. Gene Ontology enrichment analysis revealed that MARVELD1 may modulate processes associated with inhibition of tumorigenesis in colon cancer. Both upstream transcription factors and downstream functional enrichment of MARVELD1 were related to the Wnt/β-catenin signaling pathway. Overexpression of MARVELD1 inhibited the expression of β-catenin and its entry into the nucleus. MARVELD1 also inhibited the proliferation, migration, and invasion of colon cancer cells. With Wnt/β-catenin activator LiCl treatment, rescue experiments demonstrated that the role of MARVELD1 in colon cancer progression was dependent on the Wnt/β-catenin pathway. These results indicate that MARVELD1 acts as a tumor suppressor and inhibits tumorigenesis via the Wnt/β-catenin pathway.
... As its name suggests, nuclear poly(A) binding protein (PABPN1) is a predominantly nuclear poly(A) binding protein that is conserved across metazoans and has a known homologue in fission (but not budding) yeast (54,445). It is thought to transiently locate to the cytoplasm while attached to newly exported mRNAs, before being dislodged by the ribosome during the pioneer round of translation (54,363,420,(446)(447)(448). Examination by cryoimmunoelectron microscopy found little evidence of cytoplasmic PABPN1, but did find that it was associated with RNA Pol II (54). ...
Poly(A) tails affect multiple aspects of gene regulation: they help identify mRNAs for nuclear export, enhance translation efficiency, and are essential to regulating mRNA degradation rate. A poly(A) tail of 200-250 residues is thought to be uniformly added to newly synthesised mRNA and later gradually removed in the cytoplasm, allowing degradation of the mRNA itself. Previous work in the lab showed that poly(A) tail addition is not uniform; soon after serum induction, transiently expressed mRNAs are exported with long poly(A) tails, but towards the end of the transcription pulse the tails of new transcripts are much shorter. In contrast, housekeeping mRNAs consistently receive only 30-70 adenosines both before and throughout the serum response and do not appear to be gradually deadenylated. Given these controversial findings, the work presented here began by assessing the suitability of the PCR-based PAT assay for detecting differences in poly(A) tail length. This was achieved by comparing poly(A) length measurements obtained using the PAT assay with those using RNase H northern blots, which detected RNA directly. PAT assays of chromatin-associated, nucleoplasmic and cytoplasmic fractions then revealed that in NIH 3T3 cells, the poly(A) tail lengths of most mRNAs tested were determined before release from the chromatin. For the remainder of mRNAs, poly(A) tail lengths were determined in the nucleoplasm. Genome-wide analysis of poly(A) tails using adapted RNA-Seq (PQ-Seq) showed that in NIH 3T3 cells, nuclear regulation of polyadenylation was widespread and was not limited to the mRNAs previously selected for PAT. Short poly(A) tails were associated with reduced stability of transiently expressed transcripts, and it logically follows that nascent poly(A) regulation resulting in production of short-tailed transcripts at the end of the response may enhance the precision with which gene expression is controlled. Specifically, production of unstable transcripts at the end of the serum response would sharpen the peak in mature mRNA levels, limiting the time during which translation can occur. Knockdown of the mRNA encoding the CCR4-NOT deadenylase subunit, CNOT1, increased chromatin and/or nucleoplasmic poly(A) tail size for all mRNAs tested, indicating it was involved in nuclear poly(A) tail regulation. Furthermore, the magnitudes of these changes were gene-specific. Preliminary data suggested that CCR4-NOT-dependent initial poly(A) regulation may also occur in human cells. As well as increasing initial poly(A) length (and therefore presumably enhancing transcript stability), Cnot1 knockdown caused decreases in pre-mRNA levels of all mRNAs tested. Together, these data suggested that the CCR4-NOT complex may mediate mRNA homeostasis in mammalian cells. Several lines of enquiry were followed to explore the mechanism through which CNOT1 both limited initial poly(A) length and seemingly promoted mRNA production. Although the exact mechanism remained elusive, differential effects of RNAi-mediated depletion versus pharmacological inhibition of the complex’s CAF1 subunit suggested that the documented involvement of CCR4-NOT in both deadenylation and transcription elongation may have been significant. The above findings complement work from other groups showing changes to CCR4-NOT subunit levels in different physiological conditions (e.g. nutrient deprivation, B cell activation). Specifically, levels of CCR4-NOT may be adjusted to simultaneously affect both mRNA production (through promoting transcription elongation) and nuclear determination of cytoplasmic mRNA stability (through promoting nuclear deadenylation). In this way, the mammalian CCR4-NOT complex may mediate high and low mRNA turnover states according to the state of the cell.
... HPG was fluorescently labeled using click-it technology (Click-&-Go Plus 488 Labeling Kit, Product No.1314 from Click Chemistry Tools), then smFISH was performed as previously described 74 .RT-qPCR. Total cell RNA extraction, DNase I treatment, and reverse transcription were performed as described previously61 . Amplifications were performed using Power SYBR® Green PCR Master mix(Applied biosystems, REF 4367659). ...
Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrate that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two β-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by a failure of mRNA degradation after successful translation termination at the PTC. Here the author developed a single-cell reporter system to identify cell-to-cell variability of nonsense-mediated mRNA decay (NMD). This approach provides a sensitive tool to investigate cellular heterogeneity of NMD in various physiological conditions.
... En absence de PTC, la première étape de lecture de l'ARNm par le ribosome permet de retirer tous les complexes EJC fixés et conduit au remplacement de l'hétérodimère CBP20/CBP80 (CBC) par eIF4E sur la coiffe à l'extrémité 5' et de la protéine PABPN1 (pour « poly (A)-binding protein nuclear 1 ») par PABPC1 (pour « poly (A)-binding protein cytoplasmic 1 ») à l'extrémité 3'. Ces modifications autorisent de multiples étapes de traduction par de nombreux ribosomes (pour revue Rissland, 2017;Sato and Maquat, 2009). Si le ribosome rencontre un PTC respectant la règle des 50-55 nt, celui-ci sera arrêté et ne pourra pas continuer sa primo-lecture afin de déplacer les derniers complexes EJC ( Figure 29). ...
Les projets développés au cours de cette thèse ont pour objectif d’explorer l’intérêt thérapeutique d’approches antisens ciblant les transcrits d’immunoglobulines (Ig) dans le traitement du myélome multiple (MM) et d’autres gammapathies monoclonales. Cette stratégie consiste à provoquer un saut d’exon à l’aide d’oligonucléotides antisens (ASO) pour induire la synthèse d’Ig tronquées et l’apoptose des plasmocytes tumoraux (Brevet WO 2017/089359). L’effet toxique des Ig sans domaine V résulte d’une amplification incontrôlée du stress du RE et de la réponse UPR (« Unfolded Protein Response »). Nous avons montré que des traitements à l’aide d’ASO dirigés contre les ARN pré-messagers de l’Ig monoclonale induisaient une forte toxicité dans des lignées de myélome et une régression tumorale dans un modèle de xénogreffe avec des injections intratumorales d’ASO. Bien que des améliorations en terme de biodistribution in vivo de ces ASO soient nécessaires, cette approche capable de cibler spécifiquement le clone tumoral en épargnant les plasmocytes sains pourrait permettre un traitement personnalisé des patients atteints de MM. De surcroît, nous avons également observé une diminution drastique de la production d’Ig à la suite d’un traitement par un ASO générique ciblant l’exon CH1γ, aussi bien dans les LB humains stimulés ou dans des lignées de myélome; cet ASO pouvant être utilisé chez tous les patients exprimant une IgG. En parallèle, nous avons exploré les liens entre stress protéique et surveillance des ARN dans les plasmocytes. Contrairement aux données de la littérature concernant des cellules non-lymphoïdes, nous avons mis en évidence une coopération entre le mécanisme de NMD (« Nonsense-Mediated mRNA Decay ») et l’UPR (« Unfolded Protein Response ») dans les plasmocytes, qui est rendue possible grâce par une faible activation de la voie PERK de l’UPR. Cette thèse a pour but de mieux comprendre l’impact des Ig tronquées dans les cellules sécrétrices d’anticorps et le lien étroit entre survie des plasmocytes et stress protéique associé à la synthèse massive d’Ig.
... Messenger ribonucleoproteins associated with CBC are drastically remodeled during or after their export [11][12][13][14]17,18]. One peculiar event is the replacement of CBC by eukaryotic translation initiation factor 4E (eIF4E), the major cytoplasmic cap-binding protein, in a translation-independent manner [17][18][19][20][21]. eIF4E plays a crucial role during conventional protein synthesis by exploiting another scaffold protein, eIF4G, which directly associates with the eIF3 complex and recruits the 40S ribosomal subunit. ...
Selective recognition and removal of faulty transcripts and misfolded polypeptides are crucial for cell viability. In eukaryotic cells, nonsense-mediated mRNA decay (NMD) constitutes an mRNA surveillance pathway for sensing and degrading aberrant transcripts harboring premature termination codons (PTCs). NMD functions also as a post-transcriptional gene regulatory mechanism by downregulating naturally occurring mRNAs. As NMD is activated only after a ribosome reaches a PTC, PTC-containing mRNAs inevitably produce truncated and potentially misfolded polypeptides as byproducts. To cope with the emergence of misfolded polypeptides, eukaryotic cells have evolved sophisticated mechanisms such as chaperone-mediated protein refolding, rapid degradation of misfolded polypeptides through the ubiquitin–proteasome system, and sequestration of misfolded polypeptides to the aggresome for autophagy-mediated degradation. In this review, we discuss how UPF1, a key NMD factor, contributes to the selective removal of faulty transcripts via NMD at the molecular level. We then highlight recent advances on UPF1-mediated communication between mRNA surveillance and protein quality control.
... In the cytoplasm, the CBC associated with the 5 -cap structure of the mRNAs is eventually replaced by eIF4E, the major cytoplasmic cap-binding protein, in a translationindependent manner and mediated by importins ␣ and  (7,8). A recent study further showed that the CBC-to-eIF4E replacement step is regulated by the coordinated actions of UPF1 and Staufen1, a negative and positive regulator, respectively (9). ...
Newly synthesized mRNA is translated during its export through the nuclear pore complex, when its 5'-cap structure is still bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein (CBP) 80 and CBP20. Despite its critical role in mRNA surveillance, the mechanism by which CBC-dependent translation (CT) is regulated remains unknown. Here, we demonstrate that the CT initiation factor (CTIF) is tethered in a translationally incompetent manner to the perinuclear region by the DEAD-box helicase 19B (DDX19B). DDX19B hands over CTIF to CBP80, which is associated with the 5'-cap of a newly exported mRNA. The resulting CBP80-CTIF complex then initiates CT in the perinuclear region. We also show that impeding the interaction between CTIF and DDX19B leads to uncontrolled CT throughout the cytosol, consequently dysregulating nonsense-mediated mRNA decay. Altogether, our data provide molecular evidence supporting the importance of tight control of local translation in the perinuclear region.
... Translational readthrough happens due to failure to terminate translation and inserts a near-cognate tRNA for the termination codon that allows elongation to continue. This readthrough at a PTC presumably decreases the likelihood of the mRNA to undergo NMD, and in following rounds of translation of the mRNA since translational readthrough removes the exon junction complexes (EJCs) downstream of the PTC (Sato and Maquat, 2009) required for most of NMD activation (Nagy and Maquat, 1998). NMD escape also may occur due to the failure of the mRNA to degrade after PTC recognition. ...
Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrates that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two b-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by failure of mRNA degradation after successful translation termination at the PTC.
... Translational readthrough happens due to failure to terminate translation and inserts a nearcognate tRNA for the termination codon that allows elongation to continue. This readthrough at a PTC presumably decreases the likelihood of the mRNA to undergo NMD, and in following rounds of translation of the mRNA since translational readthrough removes the exon junction complexes (EJCs) downstream of the PTC (Sato and Maquat, 2009) required for most of NMD activation (Nagy and Maquat, 1998). NMD escape also may occur due to the failure of the mRNA to degrade after PTC recognition. ...
Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrate that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two β-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by a failure of mRNA degradation after successful translation termination at the PTC.
... Within the nucleus, where RBP concentrations are much higher [71], mRNPs may adopt a more linear, extended conformation [146,147], consistent with the action of RBPs to reduce cis RNA folding. After nucleocytoplasmic export, the pioneer round of translation removes proteins coating the mRNA, such as the exon junction complex, and remodels the mRNP [148]. Cytosolic mRNA structures are further modulated by translation because ribosomes greatly decompact mRNAs [37,147]. ...
Ribonucleoprotein (RNP) granules are RNA-protein assemblies involved in multiple aspects of RNA metabolism and are linked with memory, development and disease. Some RNP granules form, in part, through formation of intermolecular RNA–RNA interactions. In vitro, such trans RNA condensation occurs readily, suggesting that cells require mechanisms to modulate RNA-based condensation. Herein, we assess mechanisms of RNA condensation and how cells modulate this phenomenon. We propose that cells control RNA condensation through ATP-dependent processes, static RNA buffering, and dynamic post-translational mechanisms. Moreover, perturbations in these mechanisms can be involved in disease. This reveals multiple cellular mechanisms of kinetic and thermodynamic control to maintain the proper distribution of RNA molecules between dispersed and condensed forms.
... Likewise, recent studies showed that PABPN1 interactome contains several ribosomal proteins and general translation factors (Banerjee et al., 2019). PABPN1 is also found to regulate X-linked inhibitor of apoptosis protein mRNA translation (Davies et al., 2008) and plays a role in the initial pioneer round of translation in the cytoplasm (Ishigaki et al., 2001;Sato and Maquat, 2009). Here, we found that PABPN1 KD decreases p63a expression without affecting p63a mRNA, which is confirmed by decreased synthesis of nascent p63a protein by PABPN1 deficiency. ...
p63 is expressed from two promoters and produces two N-terminal isoforms, TAp63 and ΔNp63. Alternative splicing creates three C-terminal isoforms p63α/β/δ whereas alternative polyadenylation in coding sequence (CDS-APA) creates two more C-terminal isoforms p63γ/ε. While several transcription factors have been identified to differentially regulate the N-terminal p63 isoforms, it is unclear how the C-terminal p63 isoforms are regulated. Thus, we determined whether PABPN1, a key regulator of APA, may differentially regulate the C-terminal p63 isoforms. We found that PABPN1 deficiency increases p63γ mRNA through CDS-APA. We also found that PABPN1 is necessary for p63α translation by modulating the binding of translation initiation factors (eIF4E and eIF4G) to p63α mRNA. Moreover, we found that the p53 family, especially p63α, regulates PABPN1 transcription, suggesting that the mutual regulation between p63 and PABPN1 forms a feedback loop. Furthermore, we demonstrated that PABPN1 deficiency inhibits cell growth, which can be rescued by ectopic ΔNp63α. Finally, we showed that PABPN1 controls the terminal differentiation of HaCaT keratinocytes by modulating ΔNp63α expression. Taken together, our findings suggest that PABPN1 is a key regulator of the C-terminal p63 isoforms through CDS-APA and mRNA translation and that the p63-PABPN1 loop modulates p63 activity and the APA landscape.
... Les protéines PABPN1 sont majoritairement remplacées par PABPC1 sur les ARNm portant eIF4E (Schoenberg and Maquat, 2012). Ce changement est favorisé par le remplacement du CBC (Sato and Maquat, 2009 (Seit-Nebi et al., 2001;Song et al., 2000). Enfin, le domaine C-terminal d'eRF1 est impliqué dans l'interaction avec ses partenaires, notamment, avec le facteur eRF3 (Cheng et al., 2009). ...
La traduction est considérée comme une étape clé de l'expression des gènes permettant à la cellule de s'adapter aux variations de son environnement en réponse aux signaux internes ou externes. Des études bioinformatiques ont montrés que la moitié des ARN messagers chez l'homme portent, en amont de leur phase codante, des éléments régulateurs appelés uORF. Le laboratoire a montré qu'un défaut de terminaison de la traduction par déplétion du facteur de terminaison eRF3 modifie l'expression de gènes dont l'ARNm contient des uORF comme le gène ATF4. Cette modification se fait soit par un mécanisme de réinitiation après traduction de l'uORF soit par une augmentation de la stabilité de l'ARNm résultant d'un défaut de sa dégradation par la voie du "Nonsense-mediated mRNA Decay" (NMD). A travers leur association dans le même complexe et leur implication dans la terminaison de la traduction et la NMD, eRF3 et Upf1 contribuent à la régulation fine de l'expression des gènes. Cependant, on ne sait pas dans quelle mesure ces deux facteurs affectent la traduction et la stabilité des ARNm. Nous avons évalué la traduction par ribosome profiling et le taux de transcrits par RNA-seq dans les cellules humaines déplétées en eRF3 ou en Upf1. Ces analyses nous ont permis de dresser une carte des uORF traduites dans le transcriptome des cellules humaines HCT116. Nous avons également observé que peu de gènes cibles sont communs entre la déplétion en eRF3 ou en Upf1. Nos résultats appuient fortement l'hypothèse qu'il y a au moins deux classes de transcrits portant des uORF, l'une dont la régulation implique la terminaison de la traduction et l'autre dont la régulation implique la NMD.
... Since stop codons are generally located in the last exon of a gene, all EJCs will usually be deposited in the coding sequence of the mRNA. During translation, these EJCs are removed by the first ribosome translating the mRNA (Dostie and Dreyfuss, 2002;Sato and Maquat, 2009;Lejeune et al., 2002), so translation termination occurs in the absence of EJCs bound to the mRNA. In contrast, PTCs are frequently located upstream of one or more introns, and translation termination on PTC-containing transcripts can thus occur while one or more EJCs are still bound to the mRNA. ...
Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.
... Cette interaction moléculaire est essentielle pour l'activité hélicase d'UPF1 et induit la formation du complexe « SURF » (Kashima et al., (pour « Poly (A)-Binding Protein Cytoplasmic 1 ») qui a la capacité de se fixer à la fois sur les transcrits liés à eIF4E et à CBP80 (Chiu et al., 2004;Kashima et al., 2006). Si aucun PTC n'est rencontré, ce premier tour de la traduction favorise l'émergence d'ARNm, contenant eIF4E et PABC1, aptes à subir plusieurs étapes de traduction (Sato and Maquat, 2009). En revanche, la présence de PTC empêche ces modifications de l'ARNm. ...
Le processus aléatoire des recombinaisons V(D)J permet d’obtenir un répertoire d’anticorps (Ac) ou immunoglobulines (Ig) hautement diversifié. En revanche, le caractère imprécis des jonctions V(D)J conduit à l’apparition de décalages du cadre de lecture dans deux tiers des cas. Ainsi, la plupart des cellules B hébergent des allèles d’Ig avec des réarrangements V(D)J non-productifs au sein de leur génome. Plusieurs études incluant celles menées au laboratoire ont montré que ces allèles non-productifs sont transcrits mais subissent une régulation post-transcriptionnelle impliquant le mécanisme de dégradation des ARNm appelé NMD « Nonsense-Mediated mRNA Decay ». Cette surveillance ARN diminue ainsi le taux d’ARNm codant pour des chaînes d’Ig tronquées. En revanche, l’impact de l’épissage alternatif des transcrits d’Ig non-productifs sur la production d’Ig aberrantes reste jusqu’ici peu exploré. L’étude de ce processus appelé NAS (« Nonsense-associated Altered Splicing »), et en particulier du phénomène de saut d’exon, présente un grand intérêt car cet épissage alternatif peut permettre la synthèse d’Ig tronquées présentant des délétions internes. Les projets développés lors de cette thèse ont révélé la toxicité des chaînes d’Ig dépourvues de domaine variable (V) dans les plasmocytes, et mis en évidence l’existence d’un nouveau point de contrôle au cours de la différenciation plasmocytaire. Ce phénomène nommé TIE-checkpoint (Truncated-Ig Exclusion) conduisant à l’élimination des plasmocytes exprimant des Ig tronquées, est la conséquence d’un saut d’exon lors de l’épissage des transcrits Ig non-productifs. Pour étudier les évènements de NAS lors de l’épissage des transcrits d’Ig dans les plasmocytes, il faut par conséquent limiter l’activation du TIE-checkpoint. A l’aide d’un modèle murin présentant un exon non-sens additionnel au locus IgH, nous avons pu analyser in vivo l’épissage alternatif par « saut d’exon » des transcrits d’Ig non-productifs. En effet, l’élimination de cet exon addtionnel aboutit à la synthèse d’une chaîne d’Ig normale et non à la production de chaînes tronquées. Cette étude a été menée dans des cellules B primaires et des plasmocytes. Les résultats obtenus ont révélé que l’hypertranscription des gènes d’Ig, qui accompagne la différenciation plasmocytaire, favorise l’épissage alternatif des transcrits d’Ig non-productifs, par un phénomène de saut d’exon. Nous avons également étudié les éventuelles connexions entre le mécanisme de NMD, impliqué dans la surveillance des ARNm, et l’UPR (« Unfolded Protein Response ») permettant de réguler l’homéostasie protéique dans les plasmocytes. De façon originale, nous avons identifié une boucle de régulation positive entre les processus de surveillance ARN (NMD) et protéique (UPR, autophagie, protéasome). La mise en évidence de cette coopération dans les plasmocytes constitue un exemple unique au vue de la littérature et, aurait pour effet de limiter la synthèse d’Ig tronquées tout en autorisant la synthèse massive d’Ig. Enfin, nous avons étudié le rôle de l’épissage des transcrits d’Ig non-codants (appelés transcrits I « germinaux ») au cours du processus de CSR « Class Switch Recombination ». Cette étude a apporté des précisions sur le rôle des sites donneurs d’épissage des exons I et révélé que la reconnaissance de ces sites d’épissage module l’intensité de la transcription de la région « switch » S adjacente, et par conséquent, son accessibilité à AID « Activation-Inducedcytidine Deaminase » lors de la CSR.
... Once matured at their 3′-ends by endonucleolytic cleavage and polyadenylation that, like splicing, can produce alternative mRNA isoforms, the resulting mature mRNAs are exported from the nucleus to cytoplasm carrying the CBC, EJCs and other RNA-binding proteins (Gonatopoulos-Pournatzis and Cowling 2014;Maquat et al. 2010;Müller-McNicoll and Neugebauer 2014). In the cytoplasm, mRNAs continue to undergo dramatic remodeling of their associated proteins, including replacement of the CBC by the cytoplasmic cap-binding protein eukaryotic translation initiation factor (eIF)4E and loss of the EJCs (Maquat et al. 2010;Sato and Maquat 2009). Of particular relevance to NMD, the CBC not only remains bound to 5′-caps during mRNA export to the cytoplasm (Ishigaki et al. 2001;Lejeune et al. 2002), but it also recruits ribosomes to support the pioneer, or first, round of mRNA translation in the cytoplasm Choe et al. 2014b;Ishigaki et al. 2001;). ...
Nonsense-mediated mRNA decay (NMD), which is arguably the best-characterized translation-dependent regulatory pathway in mammals, selectively degrades mRNAs as a means of post-transcriptional gene control. Control can be for the purpose of ensuring the quality of gene expression. Alternatively, control can facilitate the adaptation of cells to changes in their environment. The key to NMD, no matter what its purpose, is the ATP-dependent RNA helicase upstream frameshift 1 (UPF1), without which NMD fails to occur. However, UPF1 does much more than regulate NMD. As examples, UPF1 is engaged in functionally diverse mRNA decay pathways mediated by a variety of RNA-binding proteins that include staufen, stem–loop-binding protein, glucocorticoid receptor, and regnase 1. Moreover, UPF1 promotes tudor-staphylococcal/ micrococcal-like nuclease-mediated microRNA decay. In this review, we first focus on how the NMD machinery recognizes an NMD target and triggers mRNA degradation. Next, we compare and contrast the mechanisms by which UPF1 functions in the decay of other mRNAs and also in microRNA decay. UPF1, as a protein polymath, engenders cells with the ability to shape their transcriptome in response to diverse biological and physiological needs.
... Though both are shuttling proteins that can move between the cytoplasm and nucleus, how and when they complete their trade-off is poorly understood. The protein landscape of a newly synthesized transcript is quite different than that of an actively translating mRNA in the cytoplasm and remodeling of many proteins must take place; for PABPN and PABPC, the first round of translation seems to promote this transformation [16,17]. The exchange between PABPN and PABPC could also be influenced by nuclear export or through passive remodeling in the cytoplasm. ...
Poly(A) tails are non-templated additions of adenosines at the 3′ ends of most eukaryotic mRNAs. In the nucleus, these RNAs are co-transcriptionally cleaved at a poly(A) site and then polyadenylated before being exported to the cytoplasm. In the cytoplasm, poly(A) tails play pivotal roles in the translation and stability of the mRNA. One challenge in studying poly(A) tails is that they are difficult to sequence and accurately measure. However, recent advances in sequencing technology, computational algorithms, and other assays have enabled a more detailed look at poly(A) tail length genome-wide throughout many developmental stages and organisms. With the help of these advances, our understanding of poly(A) tail length has evolved over the past 5 years with the recognition that highly expressed genes can have short poly(A) tails and the elucidation of the seemingly contradictory roles for poly(A)-binding protein (PABP) in facilitating both protection and deadenylation.
... Our analysis of MLN51 binding site data showed that these sites were bound before the leader EEJs ( Figure 8B). Although the EJCs are removed during the pioneer round of translation (113,114), it is possible that the interaction between MLN51 and eIF3 persists for the subsequent round of translation (11). ...
Introns in mRNA leaders are common in complex eukaryotes, but often overlooked. These introns are spliced out before translation, leaving exon-exon junctions in the mRNA leaders (leader EEJs). Our multi-omic approach shows that the number of leader EEJs inversely correlates with the main protein translation, as does the number of upstream open reading frames (uORFs). Across the five species studied, the lowest levels of translation were observed for mRNAs with both leader EEJs and uORFs (29%). This class of mRNAs also have ribosome footprints on uORFs, with strong triplet periodicity indicating uORF translation. Furthermore, the positions of both leader EEJ and uORF are conserved between human and mouse. Thus, the uORF, in combination with leader EEJ predicts lower expression for nearly one-third of eukaryotic proteins.
... Taken together, these results indicate that, in addition to the CBM directly binding CBP80, the RRM and RS domain may involve other protein-protein and/or protein-RNA interactions that control the subcellular localization of PGC-1α and/or directly stabilize its association with the CBC in cells. Notably, neither Flag-PGC-1α(WT) nor any of the variants coimmunoprecipitated with eukaryotic translation initiation factor 4E (eIF4E) (Fig. 2F, G), indicating that PGC-1α is removed from newly synthesized transcripts by the time the CBC is replaced by eIF4E and thus is not associated with the bulk of translationally active cytoplasmic mRNAs (Lejeune et al. 2002;Sato and Maquat 2009). These results are consistent with our previous observation that PGC-1α and CBP80 largely associate in the nucleus (Fig. 2C). ...
Although peroxisome proliferator-activated receptor-γ (PPARγ) coactivator 1α (PGC-1α) is a well-established transcriptional coactivator for the metabolic adaptation of mammalian cells to diverse physiological stresses, the molecular mechanism by which it functions is incompletely understood. Here we used in vitro binding assays, X-ray crystallography, and immunoprecipitations of mouse myoblast cell lysates to define a previously unknown cap-binding protein 80 (CBP80)-binding motif (CBM) in the C terminus of PGC-1α. We show that the CBM, which consists of a nine-amino-acid α helix, is critical for the association of PGC-1α with CBP80 at the 5' cap of target transcripts. Results from RNA sequencing demonstrate that the PGC-1α CBM promotes RNA synthesis from promyogenic genes. Our findings reveal a new conduit between DNA-associated and RNA-associated proteins that functions in a cap-binding protein surveillance mechanism, without which efficient differentiation of myoblasts to myotubes fails to occur.
... A l'extrémité γ' de l'ARNm, la protéine PABPN1 (pour « Poly (A)- (Chiu et al., 2004;Kashima et al., 2006). Si aucun PTC n'est rencontré, ce premier tour de la traduction favorise l'émergence d'ARNm, contenant eIF4E et PABC1, aptes à subir de multiples étapes de traduction (Sato and Maquat, 2009). ...
Le processus de recombinaison V(D)J des gènes d’immunoglobulines (Ig) est caractérisé par une grande imprécision des jonctions entre les segments variables (V), de diversité (D) et de jonction (J). Deux fois sur trois, un décalage du cadre de lecture apparaît, aboutissant à une jonction non productive dite « hors phase ». Plusieurs études ont démontré que les deux allèles productifs et non-productifs sont activement transcrits. Les transcrits matures issus des allèles non-productifs sont pris en charge par un mécanisme de surveillance des ARNm appelé NMD « Nonsense-Mediated mRNA Decay ». En dégradant efficacement les ARNm d’Ig contenant des codons non-sens, ce mécanisme prévient l’apparition des Ig tronquées au cours de l’ontogénie B. Néanmoins, aucune étude n’a jusqu’ici analysé l’impact de l’épissage alternatif des transcrits d’Ig non-productifs. Ce phénomène appelé NAS « Nonsense-associated Altered Splicing » peut conduire à une production d’Ig tronquées présentant des délétions internes du domaine variable (V).Les projets développés lors de cette thèse ont montré que la présence d’un codon non-sens, au niveau de l’exon variable (VJ) des transcrits Igκ, favorise le saut d’exon et la production de chaînes légères dépourvues de domaine variable (ΔV-κLCs). De façon intéressante, ces Ig tronquées provoquent un stress cellulaire et conduisent à l’apoptose des plasmocytes (Article 1). Ces observations ont permis d’identifier un nouveau point de contrôle agissant tardivement lors de la différenciation plasmocytaire : le TIE « Truncated-Ig Exclusion » checkpoint. Ce processus de contrôle provoque l’élimination des plasmocytes qui produisent des chaînes d’Ig tronquées. Nous avons également étudié l’épissage alternatif des transcrits d’Ig non-productifs en l’absence de TIE-checkpoint (Article 2). Cette étude a révélé que l’hypertranscription des gènes d’Ig dans les plasmocytes favorise l’épissage alternatif des transcrits d’Ig non-productifs. En utilisant un modèle d’expression forcée d’Ig tronquées, nous avons mis en évidence une coopération entre les mécanismes assurant la surveillance des ARNm (NMD) et la surveillance au niveau protéique (UPR : « Unfolded Protein Response », autophagie) (Article 3). Sur la base de ces résultats, nous avons mis au point une nouvelle approche thérapeutique qui consiste à forcer la production d’Ig tronquées en utilisant des oligonucléotides anti-sens (AON) capables de provoquer l’élimination de l’exon variable lors de l’épissage. Cette invention pourrait ouvrir des perspectives thérapeutiques pertinentes dans le traitement du Myélome Multiple et d’autres pathologies touchant les plasmocytes.
... It has been demonstrated that both translation-independent and translation-dependent remodeling mechanisms can initiate the very first translation (Chiu et al., 2004). As for translationindependent mechanism, binding of Importin β to CBP80 NLS-Importin α complex destabilizes its binding to the cap structure (Gorlich et al., 1996;Sato and Maquat, 2009). Indeed, when differentiated SH-SY5Y cells were stained with anti-importin β antibody, a punctate staining pattern was identified throughout neurites (data not shown). ...
RNA transport and regulated local translation play critically important roles in spatially restricting gene expression in neurons. Heterogeneous population of RNA granules serve as motile units to translocate, store, translate, and degrade mRNAs in the dendrites contain cis-elements and trans-acting factors such as RNA-binding proteins and microRNAs to convey stimulus-, transcript-specific local translation. Here we report a class of mRNA granules in human neuronal processes that are enriched in the nuclear cap-binding protein complex (CBC) and exon junction complex (EJC) core components, Y14 and eIF4AIII. These granules are physically associated with stabilized microtubules and are spatially segregated from eIF4E-enriched granules and P-bodies. The existence of mRNAs retaining both nuclear cap binding protein and EJC in the distal sites of neuronal processes suggests that some localized mRNAs have not yet undergone the “very first translation,” which contribute to the spatio-temporal regulation of gene expression.
... mRNPs are dynamic, and their organization changes throughout the life cycle of an mRNA [1,3,4,8]. For instance, upon entry into the cytoplasm, nuclear mRNP components are exchanged for their cytoplasmic counterparts or, as is often the case with the exonjunction complex, removed entirely [9][10][11]. Decay also changes mRNP organization. ...
Background
All mRNAs are bound in vivo by proteins to form mRNA–protein complexes (mRNPs), but changes in the composition of mRNPs during posttranscriptional regulation remain largely unexplored. Here, we have analyzed, on a transcriptome-wide scale, how microRNA-mediated repression modulates the associations of the core mRNP components eIF4E, eIF4G, and PABP and of the decay factor DDX6 in human cells.
Results
Despite the transient nature of repressed intermediates, we detect significant changes in mRNP composition, marked by dissociation of eIF4G and PABP, and by recruitment of DDX6. Furthermore, although poly(A)-tail length has been considered critical in post-transcriptional regulation, differences in steady-state tail length explain little of the variation in either PABP association or mRNP organization more generally. Instead, relative occupancy of core components correlates best with gene expression.
Conclusions
These results indicate that posttranscriptional regulatory factors, such as microRNAs, influence the associations of PABP and other core factors, and do so without substantially affecting steady-state tail length.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-017-1330-z) contains supplementary material, which is available to authorized users.
... After being processed in the nucleus, newly synthesized mRNAs are exported to the cytoplasm with their 5 0 -cap bound to the nuclear cap-binding protein complex (CBC), a heterodimer of capbinding protein 80 (CBP80, also known as nuclear cap-binding protein 1 (NCBP1)) and either CBP20 (also known as NCBP2) or NCBP3 (refs 2,3). In the cytoplasm, CBC is replaced by the cytoplasmic eukaryotic translation initiation factor 4E (eIF4E) in a translation-independent manner 4 . Both CBC and eIF4E can recruit ribosomes 5 . ...
Misfolded polypeptides are rapidly cleared from cells via the ubiquitin–proteasome system (UPS). However, when the UPS is impaired, misfolded polypeptides form small cytoplasmic aggregates, which are sequestered into an aggresome and ultimately degraded by aggrephagy. Despite the relevance of the aggresome to neurodegenerative proteinopathies, the molecular mechanisms underlying aggresome formation remain unclear. Here we show that the CTIF–eEF1A1–DCTN1 (CED) complex functions in the surveillance of either pre-existing or newly synthesized polypeptides by linking two molecular events: selective recognition and aggresomal targeting of misfolded polypeptides. These events are accompanied by CTIF sequestration into the aggresome, preventing the additional synthesis of misfolded polypeptides from mRNAs bound by nuclear cap-binding complex. These events render cells more resistant to apoptosis induced by proteotoxic stresses. Collectively, our data provide compelling evidence for a previously unappreciated protein surveillance pathway and a regulatory gene expression network for coping with misfolded polypeptides.
... EJCs facilitate the recruitment of ribosomes to spliced mRNAs and translation initiation (68)(69)(70)(71)(72)(73)(74)(75). Although EJCs are subsequently removed by elongating ribosomes (76)(77)(78), spliced mRNAs are translated to produce several times more protein per mRNA than in unspliced mRNAs that have an identical primary sequence but lack EJCs (79,80). Since the mRNP structure could affect the rate of mRNA degradation, it was important to determine whether splicing affects the sensitivity of an mRNA to Vhs. ...
Importance:
Most mammalian mRNAs are spliced. In contrast, of the more than 80 mRNAs encoded by herpes simplex virus type 1 (HSV-1), only five are spliced. In addition, synthesis of the immediate-early protein ICP27 causes partial inhibition of pre-mRNA splicing, with the resultant accumulation of both spliced and unspliced versions of some mRNAs in the cytoplasm. A common perception is that HSV-1 infection necessarily inhibits expression of spliced mRNAs. In contrast, this study demonstrates two instances in which pre-mRNA splicing actually enhances the synthesis of proteins from mRNAs during HSV-1 infections. Specifically, splicing stabilized an mRNA against degradation by copies of the Vhs endoribonuclease from infecting virions, and greatly enhanced the amount of protein synthesized from spliced mRNAs at late times after infection. The data suggest that splicing, and the resultant presence of exon junction complexes on an mRNA, may play an important role in gene expression during HSV-1 infections.
... [39][40][41] After export to the cytoplasm, this complex is replaced by eIF4E in a key mRNP remodeling step. 42,43 Similarly, eIF4E2 (also known as 4EHP) can bind the 5 0 cap in the cytoplasm, but it AAAAAAA eIF4G eIF4E PABP FIGURE 2 | The closed loop model. By binding simultaneously to eIF4E and PABP, which in turn bind the 5 0 cap and 3 0 poly(A) tail, eIF4G forms a protein bridge that brings the two transcript ends together and allows regulatory information (such as deadenylation) to be transmitted from the 3 0 to 5 0 end of an mRNA. ...
In a eukaryotic cell, each messenger RNA (mRNA) is bound to a variety of proteins to form an mRNA–protein complex (mRNP). Together, these proteins impact nearly every step in the life cycle of an mRNA and are critical for the proper control of gene expression. In the cytoplasm, for instance, mRNPs affect mRNA translatability and stability and provide regulation of specific transcripts as well as global, transcriptome‐wide control. mRNPs are complex, diverse, and dynamic, and so they have been a challenge to understand. But the advent of high‐throughput sequencing technology has heralded a new era in the study of mRNPs. Here, I will discuss general principles of cytoplasmic mRNP organization and regulation. Using microRNA‐mediated repression as a case study, I will focus on common themes in mRNPs and highlight the interplay between mRNP composition and posttranscriptional regulation. mRNPs are an important control point in regulating gene expression, and while the study of these fascinating complexes presents remaining challenges, recent advances provide a critical lens for deciphering gene regulation. WIREs RNA 2017, 8:e1369. doi: 10.1002/wrna.1369
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Main conclusion
Nonsense-mediated mRNA decay in eukaryotes is vital to cellular homeostasis. Further knowledge of its putative role in plant RNA metabolism under stress is pivotal to developing fitness-optimizing strategies.
Abstract
Nonsense-mediated mRNA decay (NMD), part of the mRNA surveillance pathway, is an evolutionarily conserved form of gene regulation in all living organisms. Degradation of mRNA-bearing premature termination codons and regulation of physiological RNA levels highlight NMD’s role in shaping the cellular transcriptome. Initially regarded as purely a tool for cellular RNA quality control, NMD is now considered to mediate various aspects of plant developmental processes and responses to environmental changes. Here we offer a basic understanding of NMD in eukaryotes by explaining the concept of premature termination codon recognition and NMD complex formation. We also provide a detailed overview of the NMD mechanism and its role in gene regulation. The potential role of effectors, including ABCE1, in ribosome recycling during the translation process is also explained. Recent reports of alternatively spliced variants of corresponding genes targeted by NMD in Arabidopsis thaliana are provided in tabular format. Detailed figures are also provided to clarify the NMD concept in plants. In particular, accumulating evidence shows that NMD can serve as a novel alternative strategy for genetic manipulation and can help design RNA-based therapies to combat stress in plants. A key point of emphasis is its function as a gene regulatory mechanism as well as its dynamic regulation by environmental and developmental factors. Overall, a detailed molecular understanding of the NMD mechanism can lead to further diverse applications, such as improving cellular homeostasis in living organisms.
In non-polarized cells, nonsense-mediated mRNA decay (NMD) generally begins during the translation of newly synthesized mRNAs after the mRNAs are exported to the cytoplasm. Binding of the FMRP translational repressor to UPF1 on NMD targets mainly inhibits NMD. However, in polarized cells like neurons, FMRP additionally localizes mRNAs to cellular projections. Here, we review the literature and evaluate available transcriptomic data to conclude that, in neurons, the translation of physiologic NMD targets bound by FMRP is partially inhibited until the mRNAs localize to projections. There, FMRP displacement in response to signaling induces a burst in protein synthesis followed by rapid mRNA decay.
In eukaryotic cells, RNAs transcribed by RNA polymerase-II receives the modification at the 5’ end. This structure is called as the cap structure. The cap structure has a fundamental role for translation initiation by recruiting eukaryotic translation initiation factor 4F(eIF4F). The other important mediator of the cap structure is a nuclear cap binding protein complex (CBC). CBC consists of two proteins, which are renamed as NCBP1 and NCBP2 (previously called as CBP80/NCBP and CBP20/NIP1, respectively). This review article discusses the multiple roles CBC mediates and co-ordinates in several gene expression steps in eukaryotes.
Mammalian poly A-binding proteins (PABPs) are highly conserved multifunctional RNA-binding proteins primarily involved in the regulation of mRNA translation and stability, of which PABPC1 is considered a central regulator of cytoplasmic mRNA homing and is involved in a wide range of physiological and pathological processes by regulating almost every aspect of RNA metabolism. Alterations in its expression and function disrupt intra-tissue homeostasis and contribute to the development of various tumors. There is increasing evidence that PABPC1 is aberrantly expressed in a variety of tumor tissues and cancers such as lung, gastric, breast, liver, and esophageal cancers, and PABPC1 might be used as a potential biomarker for tumor diagnosis, treatment, and clinical application in the future. In this paper, we review the abnormal expression, functional role, and molecular mechanism of PABPC1 in tumorigenesis and provide directions for further understanding the regulatory role of PABPC1 in tumor cells.
Among the more than 170 known RNA modifications, methylation modification is the most frequent and well‐studied. Depending on where the methylation occurs, RNA methylation can be classified as N6‐methyladenosine, N1‐methyladenosine, 5‐methylcytosine, N7‐methylguanosine, and others. The methylation of RNA is constantly and dynamically modified in the complex microenvironment by methyltransferases, demethylases, and methylation reading proteins. These changes affect the proliferation and differentiation of immune cells as well as their effector activities by affecting RNA location, activity, stability, and translation efficiency. This review outlines how diverse RNA methylation alterations affect immune cell development and biological activity, as well as the role of RNA methylation in health and disease, to provide a molecular basis for future immunotherapies.
Quality control of mRNA represents an important regulatory mechanism for gene expression in eukaryotes. One component of this quality control is the nuclear retention and decay of misprocessed RNAs. Previously, we demonstrated that mature mRNAs containing a 5’ splice site (5’SS) motif, which is typically found in misprocessed RNAs such as intronic polyadenylated (IPA) transcripts, are nuclear retained and degraded. Here we demonstrate that these transcripts require the zinc finger protein ZFC3H1 for their decay and nuclear retention into nuclear speckles. Furthermore, we find that U1-70K, a component of the U1 snRNP spliceosomal complex, is also required for their nuclear retention and likely functions in the same pathway as ZFC3H1. Finally, we show that the disassembly of nuclear speckles impairs the nuclear retention of mRNAs with 5’SS motifs. Together, our results suggest a model where mRNAs with 5’SS motifs are recognized by U1 snRNP, which then acts with ZFC3H1 to both promote their decay and prevent nuclear export of these mRNAs by sequestering them in nuclear speckles. Our results highlight a splicing independent role of U1 snRNP and indicate that it works in conjunction with ZFC3H1 in preventing the nuclear export of misprocessed mRNAs.
Nonsense-mediated mRNA decay (NMD), a cellular RNA quality system, has been shown to be an ancestral form of cellular antiviral response that can restrict viral infection by targeting viral RNA for degradation or other various mechanisms. In support to this hypothesis, emerging evidences unraveled that viruses have evolved numerous mechanisms to circumvent or modulate the NMD pathway to ensure unhindered replication within the host cell. In this study, we investigated the potential interplay between the cellular NMD pathway and rotavirus (RV). Our data suggested that rotavirus infection resulted in global inhibition of NMD pathway by down regulating the expression of UPF1 in a strain independent manner. UPF1 expression was found to be regulated at the post-transcriptional level by ubiquitin-proteasome mediated degradation pathway. Subsequent studies revealed rotaviral non-structural protein 5 (NSP5) associates with UPF1 and promotes its cullin-dependent proteasome mediated degradation. Furthermore, ectopic expression of UPF1 during RV infection resulted in reduced expression of viral proteins and viral RNAs leading to diminished production of infective rotavirus particles, suggesting the anti-rotaviral role of UPF1. Finally, the delayed degradation kinetics of transfected rotaviral RNA in UPF1 and UPF2 depleted cells and the association of UPF1 and UPF2 with viral RNAs suggested that NMD targets rotaviral RNAs for degradation. Collectively, the present study demonstrates the antiviral role of NMD pathway during rotavirus infection and also reveals the underlying mechanism by which rotavirus overwhelms NMD pathway to establish successful replication.
In mammals, two different messenger ribonucleoproteins (mRNPs) serve as templates for protein synthesis. Newly synthesized CBP80‐CBP20 (CBC)‐bound mRNPs initially undergo a pioneer round of translation (Maquat et al., 2010). One purpose of this round is to ensure the quality of gene expression, as exemplified by nonsense‐mediated mRNA decay (NMD). NMD functions to eliminate mRNAs that prematurely terminate translation, and also contributes to proper gene control, and it targets CBC‐bound mRNPs (Sato et al., 2008; Isken et al., 2008). CBC‐bound mRNPs are remodeled to eIF4E‐bound mRNPs as a consequence of the pioneer round of translation and also independently of translation (Sato & Maquat, 2009). eIF4E‐bound mRNPs support the bulk of cellular protein synthesis and are the primary targets of mRNA decay mechanisms that conditionally regulate gene expression, such as Staufen1‐mediated mRNA decay (SMD) (Gong et al., 2009). Mechanistic aspects of NMD will be discussed, including how CBP80, which is acquired by the 5′ caps of newly synthesized transcripts, promotes NMD at multiple steps by promoting specific mRNP rearrangements (Hwang et al., 2010). Mechanistic aspects of SMD will also be described, including how Staufen1‐binding sites form not only by intramolecular base‐pairing within an mRNA 3′‐untranslated region but also by intermolecular basepairing between the Alu element of an mRNA 3‐untranslated region and a partially complementary Alu element within a long noncoding RNA (Gong & Maquat, 2011).
Nonsense-mediated mRNA decay (NMD) is one of the best characterized and most evolutionarily conserved cellular quality control mechanisms. Although NMD was first found to target one-third of mutated, disease-causing mRNAs, it is now known to also target ~10% of unmutated mammalian mRNAs to facilitate appropriate cellular responses — adaptation, differentiation or death — to environmental changes. Mutations in NMD genes in humans are associated with intellectual disability and cancer. In this Review, we discuss how NMD serves multiple purposes in human cells by degrading both mutated mRNAs to protect the integrity of the transcriptome and normal mRNAs to control the quantities of unmutated transcripts.
Ribonucleic acid (RNA) homeostasis is dynamically modulated in response to changing physiological conditions. Tight regulation of RNA abundance through both transcription and degradation determines the amount, timing, and location of protein translation. This balance is of particular importance in neurons, which are among the most metabolically active and morphologically complex cells in the body. As a result, any disruptions in RNA degradation can have dramatic consequences for neuronal health. In this chapter, we will first discuss mechanisms of RNA stabilization and decay. We will then explore how the disruption of these pathways can lead to neurodegenerative disease.
Binding of a long series of mono- and dinucleotide analogues of the 7-methylguanosine containing 5'-mRNA-cap to human protein translation initiation factor eIF4E has been investigated by means of fluorescence. A new methodological approach in gathering and analysis of the fluorescence data provided us with very accurate values of the association equilibrium constant K and normalized, maximal quenching of the protein fluorescence Delta F-max, during titration of eIF4E by various cap-analogues. The results confirm participation of at least two conserved tryptophan residues of eIF4E in interaction with 7-methylguanine, as has been described recently for murine eIF4E, complexed with 7-methyl-GDP in crystal (Marcotrigiano et al., 1997, Cell 89, 951), and for yeast eIF4E, complexed with the same ligand in solution (Matsuo et al., 1997, Nature Struct. Biol. 4, 717). On the other hand binding by eIF4E of unmethylated guanine nucleotides and N-2,N-2,7-trimrthylguanine containing nucleotides differ substantially from the way of binding of the regular mRNA-cap. Influence of the structural features of the cap-analogues, especially the type of the second nucleoside in the dinucleotide caps, on their association with eIF4E and biological activities in in vitro protein translation systems has been discussed in light of the known structures of the eIF4E-7-methyl-GDP complexes in crystal and solution.
Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs. Premature termination codons in the -globin mRNA cause the common recessive form of -thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay. We demonstrate that cells distinguish a premature termination codon within the -globin mRNA from the physiological translation termination codon by a two-step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag. In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA. This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.
Among the different cellular surveillance mechanisms in charge to prevent production of faulty gene products, nonsense-mediated mRNA decay (NMD) represents a translation-dependent posttranscriptional process that selectively recognizes and degrades mRNAs whose open reading frame (ORF) is truncated by a premature translation termination codon (PTC, also called "nonsense codon"). In doing so, NMD protects the cell from accumulating C-terminally truncated proteins with potentially deleterious functions. Transcriptome profiling of NMD-deficient yeast, Drosophila, and human cells revealed that 3-10% of all mRNA levels are regulated (directly or indirectly) by NMD, indicating an important role of NMD in gene regulation that extends beyond quality control [J. Rehwinkel, J. Raes, E. Izaurralde, Nonsense-mediated mRNA decay: Target genes and functional diversification of effectors, Trends Biochem. Sci. 31 (2006) 639-646.[1]]. In this review, we focus on recent results from different model organisms that indicate an evolutionarily conserved mechanism for PTC identification.
The eukaryotic translation initiation factor 4G (eIF4G) proteins play a critical role in the recruitment of the translational machinery to mRNA. The eIF4Gs are phosphoproteins. However, the location of the phosphorylation sites, how phosphorylation of these proteins is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established. In this report, two-dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residues is altered by serum and mitogens. Phosphopeptides resolved by this method were mapped to the C–terminal one-third of the protein. Mass spectrometry and mutational analyses identified the serum-stimulated phosphorylation sites in this region as serines 1108, 1148 and 1192. Phosphoinositide–3–kinase (PI3K) inhibitors and rapamycin, an inhibitor of the kinase FRAP/mTOR (FKBP12–rapamycin-associated protein/mammalian target of rapamycin), prevent the serum-induced phosphorylation of these residues. Finally, the phosphorylation state of N–terminally truncated eIF4GI proteins acquires resistance to kinase inhibitor treatment. These data suggest that the kinases phosphorylating serines 1108, 1148 and 1192 are not directly downstream of PI3K and FRAP/mTOR, but that the accessibility of the C–terminus to kinases is modulated by this pathway(s).
Eukaryotic initiation factor 4A (eIF4A) is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5′ proximal secondary structure of eukaryotic mRNAs to facilitate attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors (eIF4E and eIF4G). Two isoforms of eIF4A, eIF4AI and eIF4AII, which are encoded by two different genes, are functionally indistinguishable. A third member of the eIF4A family, eIF4AIII, whose human homolog exhibits 65% amino acid identity to human eIF4AI, has also been cloned from
Xenopus
and tobacco, but its function in translation has not been characterized. In this study, human eIF4AIII was characterized biochemically. While eIF4AIII, like eIF4AI, exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, it fails to substitute for eIF4AI in an in vitro-reconstituted 40S ribosome binding assay. Instead, eIF4AIII inhibits translation in a reticulocyte lysate system. In addition, whereas eIF4AI binds independently to the middle and carboxy-terminal fragments of eIF4G, eIF4AIII binds to the middle fragment only. These functional differences between eIF4AI and eIF4AIII suggest that eIF4AIII might play an inhibitory role in translation under physiological conditions.
Export of messenger RNA from the transcription site in the nucleus and mRNA targeting to the translation site in the cytoplasm are key regulatory processes in protein synthesis. In yeast, the mRNA-binding proteins Nab2p and Nab4p/Hrp1p accompany transcripts to their translation site, where the karyopherin Kap104p mediates both their dissociation from the mRNA and their transport back into the nucleus. We found that Kap104p localized to the distal bud tip and the bud neck during cell division, resulting in a localized release of translation-competent mRNA and increased protein synthesis in the emerging daughter cell. Temporally and spatially coordinated localization of Kap104p is a new mechanism for the asymmetric distribution of protein synthesis in dividing cells.
The 5' cap structure m7GpppN (where N is any nucleotide) is a ubiquitous feature of cellular eukaryotic mRNAs. The cap is multifunctional as it is involved in translation, nucleocytoplasmic transport, splicing, and stabilization of mRNA against 5' exonucleolytic degradation. The cap binding protein, eukaryotic initiation factor 4E (eIF-4E), is a translation initiation factor that binds to the cap structure and is part of a complex (eIF-4F) that promotes mRNA binding to ribosomes. Overexpression of eIF-4E in fibroblasts results in cell transformation. To test the hypothesis that some of the biological effects of eIF-4E might be effected by a nuclear function, we determined the cellular distribution of eIF-4E. By means of indirect immunofluorescence experiments using polyclonal and monoclonal antibodies against eIF-4E as well as transfected epitope-tagged eIF-4E, we demonstrate that a fraction of eIF-4E localizes to the nucleus. These results suggest that eIF-4E is also involved in a nuclear function.
Rapid degradation of c-fos proto-oncogene mRNA is crucial for transient c-fos gene expression. Experiments were performed to investigate the cellular mechanisms responsible for the extremely short half-life of human c-fos mRNA in growth-factor-stimulated fibroblasts. These experiments demonstrate the existence of two distinct cellular pathways for rapid c-fos mRNA degradation. Each of these pathways recognizes a different, functionally independent instability determinant within the c-fos transcript. One instability determinant, which is located within the c-fos 3'-untranslated region, is a 75-nucleotide AU-rich segment. Insertion of this element into beta-globin mRNA markedly reduces the half-life of that normally long-lived message. Nevertheless, specific deletion of the AU-rich element from c-fos mRNA has little effect on the transcript's cytoplasmic half-life due to the presence of the other c-fos instability determinant, which is located in the protein-coding segment of the c-fos message. Examination of mRNA decay in cells treated with transcription inhibitors indicates that one c-fos mRNA degradation pathway is dependent on RNA synthesis, whereas the other is not.
Small nuclear ribonucleoprotein particles (snRNPs) of the U-snRNP class from Ehrlich ascites tumor cells were purified in a one-step procedure by affinity chromatography with antibodies specific for 2,2,7-trimethylguanosine (m23.2.7G), which is part of the 5'-terminal cap structure of snRNAs U1-U5. Antibody-bound snRNPs are desorbed from the affinity column by elution with excess nucleoside m23.2.7G; this guarantees maintenance of their native structure. The snRNPs U1, U2, U4, U5 and U6 can be recovered quantitatively from nuclear extracts by this procedure. Co-isolation of U6 snRNP must be due to interactions between this and other snRNPs, as anti-m23.2.7G antibodies do not react with deproteinized U6 snRNA. We have so far defined nine proteins of approximate mol. wts. 10 000, 12 000, 13 000, 16 000, 21 000, 28 000, 32 000, 34 000 and 75 000. Purified snRNPs react with anti-(U1)RNP and with anti-Sm antisera from patients with mixed connective tissue disease and from MRL/l mice. As determined by the protein blotting technique, six of the snRNP polypeptides, characterized by apparent mol. wts. 13 000, 16 000, 21 000, 28 000, 34 000 and 75 000, bear antigenic determinants for one or the other of the above autoantibody classes. This suggests strongly that the U-snRNPs produced by the procedure described here are indeed representative of the snRNPs in the cell. With highly purified snRNPs available, investigation of possible enzymic functions of the particles may now be undertaken.
Ser-53 has previously been considered the major phosphorylation site in eukaryotic initiation factor (eIF)-4E, and this appeared
to be supported by studies using a S53A mutant. Recently, however, several lines of evidence have indicated that Ser-53 might
not be the true phosphorylation site. This prompted us to re-examine the phosphorylation site in eIF-4E using factor purified
from P-labeled, serum-treated Chinese hamster ovary cells. Isoelectric focusing and phosphoamino acid analysis indicated the existence
of a single phosphorylated serine. Edman degradation of the major radiolabeled tryptic product from P-labeled eIF-4E showed that the phosphorylated site was positioned three residues from the N terminus of this peptide. There
are three serines in the sequence of eIF-4E that are three residues away from a tryptic cleavage site (i.e. lysine or arginine). P-Labeled eIF-4E was digested with trypsin, Lys-C, or trypsin followed by Glu-C and subjected to two-dimensional mapping;
the data obtained eliminated two of these potential sites, leaving Ser-209. Comigration of the synthetic peptide SGS(P)TTK with the radiolabeled tryptic product on (i) reverse-phase chromatography and (ii) two-dimensional mapping at different
pH values confirmed that Ser-209 is the major phosphorylation site in eIF-4E in serum-stimulated Chinese hamster ovary cells.
In vertebrates, a nuclear cap-binding complex (CBC) formed by two cap- binding proteins, CBP20 and CBP80, is involved in several steps of RNA metabolism, including pre-mRNA splicing and nuclear export of some RNA polymerase II-transcribed U snRNAs. The CBC is highly conserved, and antibodies against human CBP20 cross-react with the CBP20 counterpart in the dipteran Chironomus tentans. Using immunoelectron microscopy, the in situ association of CBP20 with a specific pre-mRNP particle, the Balbiani ring particle, has been analyzed at different stages of pre-mRNA synthesis, maturation, and nucleo-cytoplasmic transport. We demonstrate that CBP20 binds to the nascent pre-mRNA shortly after transcription initiation, stays in the RNP particles after splicing has been completed, and remains attached to the 5' domain during translocation of the RNP through the nuclear pore complex (NPC). The rapid association of CBP20 with nascent RNA transcripts in situ is consistent with the role of CBC in splicing, and the retention of CBC on the RNP during translocation through the NPC supports its proposed involvement in RNA export.
The complex of importin-alpha and -beta is essential for nuclear protein import. It binds the import substrate in the cytosol, and the resulting trimeric complex moves through the nuclear pores, probably as a single entity. Importin-alpha provides the nuclear localization signal binding site, importin-beta the site of initial docking to the pore. Here we show that the conserved, basic N-terminus of importin-alpha is sufficient for importin-beta binding and essential for protein import. The fusion product of this 41 amino acid domain to a heterologous protein if transported into the nucleus in the same way as full-length importin-alpha itself. Transport is dependent on importin-beta but competed by importin-alpha. As no additional part of importin-alpha is needed for translocation, the movement which drives the import substrate complex into the nucleus appears to be generated between importin-beta and structures of the nuclear pore. The domain that binds to importin-beta appears to confer import only, but not re-export out of the nucleus, suggesting that the return of importin-alpha into the cytoplasm is not a simple reversal of its entry.
Targeting of most nuclear proteins to the cell nucleus is initiated by interaction between the classical nuclear localization signals (NLSs) contained within them and the importin NLS receptor complex. We have recently delineated a novel 38 amino acid transport signal in the hnRNP A1 protein, termed M9, which confers bidirectional transport across the nuclear envelope. We show here that M9-mediated nuclear import occurs by a novel pathway that is independent of the well-characterized, importin-mediated classical NLS pathway. Additionally, we have identified a specific M9-interacting protein, termed transportin, which binds to wild-type M9 but not to transport-defective M9 mutants. Transportin is a 90 kDa protein, distantly related to importin beta, and we show that it mediates the nuclear import of M9-containing proteins. These findings demonstrate that there are at least two receptor-mediated nuclear protein import pathways. Furthermore, as hnRNP A1 likely participates in mRNA export, it raises the possibility that transportin is a mediator of this process as well.
The cap structure, m7GpppN, is present at the 5′-end of all eukaryotic cellular (except organellar) mRNAs. Initiation of translation is mediated
by the multisubunit initiation factor eIF4F, which binds the cap structure via its eIF4E subunit and facilitates the binding
of mRNA to ribosomes. Here, we used recombinant proteins to reconstitute the cap recognition activity of eIF4F in vitro. We demonstrate that the interaction of eIF4E with the mRNA 5′-cap structure is dramatically enhanced by eIF4G, as determined
by a UV-induced cross-linking assay. Furthermore, assembly of the eIF4F complex at the cap structure, as well as ATP hydrolysis,
is shown to be a requisite for the cross-linking of another initiation factor, eIF4B, to the cap structure. In addition, the
stimulatory effect of eIF4G on the cap recognition of eIF4E is inhibited by the translational repressor, 4E-BP1. These results
suggest that eIF4E initially interacts with the mRNA cap structure as part of the eIF4F complex.
Binding of iron regulatory proteins (IRPs) to IREs located in proximity to the cap structure of ferritin H- and L-chain mRNAs blocks ferritin synthesis by preventing the recruitment of the small ribosomal subunit to the mRNA. We have devised a novel procedure to examine the assembly of translation initiation factors (eIFs) on regulated mRNAs. Unexpectedly, we find that the cap binding complex eIF4F (comprising eIF4E, eIF4G, and eIF4A) assembles even when IRP-1 is bound to the cap-proximal IRE. This assembly is futile, because bridging interactions between eIF4F and the small ribosomal subunit cannot be established in the presence of IRP-1. Our findings provide insight into translational control by an mRNA binding protein at the level of translation initiation factors and uncover a key regulatory step in iron homeostasis.
We have identified multiple distinct splicing enhancer elements within protein-coding sequences of the constitutively spliced
human β-globin pre-mRNA. Each of these highly conserved sequences is sufficient to activate the splicing of a heterologous
enhancer-dependent pre-mRNA. One of these enhancers is activated by and binds to the SR protein SC35, whereas at least two
others are activated by the SR protein SF2/ASF. A single base mutation within another enhancer element inactivates the enhancer
but does not change the encoded amino acid. Thus, overlapping protein coding and RNA recognition elements may be coselected
during evolution. These studies provide the first direct evidence that SR protein-specific splicing enhancers are located
within the coding regions of constitutively spliced pre-mRNAs. We propose that these enhancers function as multisite splicing
enhancers to specify 3′ splice-site selection.
In an attempt to further understand how nuclear events (such as gene expression, nuclear import/export, and cell cycle checkpoint
control) might be subject to regulation by extracellular stimuli, we sought to identify nuclear activities under growth factor
control. Using a sensitive photoaffinity labeling assay that measured [α-32P]GTP incorporation into nuclear proteins, we identified the 20-kDa subunit of the nuclear cap-binding complex (CBC) as a
protein whose binding activity is greatly enhanced by the extracellular stimulation of serum-arrested cells. The CBC represents
a 20- and 80-kDa heterodimer (the subunits independently referred to as CBP20 and CBP80, respectively) that binds the 7-methylguanosine
cap on RNAs transcribed by RNA polymerase II. This binding facilitates precursor messenger RNA splicing and export. We have
demonstrated that the [α-32P]GTP incorporation into CBP20 was correlated with an increased ability of the CBC to bind capped RNA and have used the [α-32P]GTP photoaffinity assay to characterize the activation of the CBC in response to growth factors. We show that the CBC is
activated by heregulin in HeLa cells and by nerve growth factor in PC12 cells as well as during the G1/S phase of the cell cycle and when cells are stressed with UV irradiation. Additionally, we show that cap-dependent splicing
of precursor mRNA, a functional outcome of CBC activation, can be catalyzed by growth factor addition to serum-arrested cells.
Taken together, these data identify the CBC as a nuclear target for growth factor-coupled signal transduction and suggest
novel mechanisms by which growth factors can influence gene expression and cell growth.
The mammalian GTP-binding protein GSPT, whose carboxyl-terminal sequence is homologous to the eukaryotic elongation factor
EF1α, binds to the polypeptide chain releasing factor eRF1 to function as eRF3 in the translation termination. The amino-terminal
domain of GSPT was, however, not required for the binding. Search for other GSPT-binding proteins in yeast two-hybrid screening
system resulted in the identification of a cDNA encoding polyadenylate-binding protein (PABP), whose amino terminus is associating
with the poly(A) tail of mRNAs presumably for their stabilization. The interaction appeared to be mediated through the carboxyl-terminal
domain of PABP and the amino-terminal region of GSPT. Interestingly, multimerization of PABP with poly(A), which is ascribed
to the action of its carboxyl-terminal domain, was completely inhibited by the interaction with the amino-terminal domain
of GSPT. These results indicate that GSPT/eRF3 may play important roles not only in the termination of protein synthesis but
also in the regulation of mRNA stability. Thus, the present study is the first report showing that GSPT/eRF3 carries the translation
termination signal to 3′-poly(A) tail ubiquitously present in eukaryotic mRNAs.
Poly(A)-binding protein II (PABP2) is an abundant nuclear protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In the cytoplasm, a distinct protein (PABP1) binds to poly(A) tails and participates in mRNA translation and stability. How cytoplasmic PABP1 substitutes for nuclear PABP2 is still unknown. Here we report that PABP2 shuttles back and forth between nucleus and cytoplasm by a carrier-mediated mechanism. A potential novel type of nuclear localization signal exists at the C-terminus of the protein, a domain that is highly enriched in methylated arginines. PABP2 binds directly to transportin in a RanGTP-sensitive manner, suggesting an involvement of this transport receptor in mediating import of the protein into the nucleus. Although PABP2 is small enough to diffuse passively through the nuclear pores, protein fusion experiments reveal the existence of a facilitated export pathway. Accordingly, no transport of PABP2 to the cytoplasm occurs at 4 degrees C. In contrast, export of PABP2 continues in the absence of transcription, indicating that transport to the cytoplasm is independent of mRNA traffic. Thus, rather than leaving the nucleus as a passive passenger of mRNAs, the data suggest that PABP2 interacts with the nuclear export machinery and may therefore contribute to mRNA transport.
In vertebrates, a nuclear cap-binding complex (CBC) formed by two cap- binding proteins, CBP20 and CBP80, is involved in several steps of RNA metabolism, including pre-mRNA splicing and nuclear export of some RNA polymerase II-transcribed U snRNAs. The CBC is highly conserved, and antibodies against human CBP20 cross-react with the CBP20 counterpart in the dipteran Chironomus tentans. Using immunoelectron microscopy, the in situ association of CBP20 with a specific pre-mRNP particle, the Balbiani ring particle, has been analyzed at different stages of pre-mRNA synthesis, maturation, and nucleo-cytoplasmic transport. We demonstrate that CBP20 binds to the nascent pre-mRNA shortly after transcription initiation, stays in the RNP particles after splicing has been completed, and remains attached to the 5' domain during translocation of the RNP through the nuclear pore complex (NPC). The rapid association of CBP20 with nascent RNA transcripts in situ is consistent with the role of CBC in splicing, and the retention of CBC on the RNP during translocation through the NPC supports its proposed involvement in RNA export.
Poly (A) tails are found at the 3′ ends of almost all eukaryotic mRNAs. They are bound by two different poly (A) binding proteins, PABPC in the cytoplasm and PABPN1 in the nucleus. PABPC functions in the initiation of translation and in the regulation of mRNA decay. In both functions, an interaction with the m7G cap at the 5′ end of the message plays an important role. PABPN1 is involved in the synthesis of poly (A) tails, increasing the processivity of poly (A) polymerase and contributing to defining the length of a newly synthesized poly (A) tail.
Nonsense-mediated decay (NMD) eliminates mRNAs that prematurely terminate translation. We used antibody to the nuclear cap binding protein CBP80 or its cytoplasmic counterpart eIF4E to immunopurify RNP containing nonsense-free or nonsense-containing transcripts. Data indicate that NMD takes place in association with CBP80. We defined other components of NMD-susceptible mRNP as CBP20, PABP2, eIF4G, and the NMD factors Upf2 and Upf3. Consistent with the dependence of NMD on translation, the NMD of CBP80-bound mRNA is blocked by cycloheximide or suppressor tRNA. These findings provide evidence that translation can take place in association with CBP80. They also indicate that CBP80-bound mRNA undergoes a “pioneer” round of translation, before CBP80-CBP20 are replaced by eIF4E, and Upf2 and Upf3 proteins dissociate from upstream of exon-exon junctions.
The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5'-triphosphate (m(7)GTP) and P-3-7-methylguanosine-P-3-adenosine-5',5'-triphosphate (m(7)GpppA)] were determined at 2.0 Angstrom resolution (where 1 Angstrom = 0.1 nm). The flexibility of the C-terminal loop region of eIF4E complexed with m(7)GTP was significantly reduced when complexed with m(7)GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch on/off of eIF4E-binding protein-eIF4E phosphorylation.
The binding of capped RNAs to the cap-binding complex (CBC) in the nucleus, and their dissociation from the CBC in the cytosol, represent essential steps in RNA processing. Here we show how the nucleocytoplasmic transport proteins importin-alpha and importin-beta have key roles in regulating these events. As a first step toward understanding the molecular basis for this regulation, we determined a 2.2-A resolution X-ray structure for a CBC-importin-alpha complex that provides a detailed picture for how importin-alpha binds to the CBP80 subunit of the CBC. Through a combination of biochemical studies, X-ray crystallographic information and small-angle scattering experiments, we then determined how importin-beta binds to the CBC through its CBP20 subunit. Together, these studies enable us to propose a model describing how importin-beta stimulates the dissociation of capped RNA from the CBC in the cytosol following its nuclear export.
Exon junction complexes (EJCs) are deposited onto mRNAs during splicing, serve as positional landmarks for the intron exon structure of genes, and direct posttranscriptional processes in the cytoplasm. EJC removal and recycling by translation are ill understood and have been attributed to ribosomal passage. This work identifies the ribosome-associated protein PYM as an EJC disassembly factor and defines its mechanism of function. Whereas EJC assembly intermediates are resistant to PYM, fully assembled EJCs are dissociated from spliced mRNAs by PYM. This disassembly involves PYM binding to the EJC proteins MAGOH-Y14. PYM overexpression in cells disrupts EJC association with spliced mRNA and inhibits nonsense-mediated mRNA decay. In cells depleted of PYM, EJCs accumulate on spliced mRNAs and EJC protein recycling is impaired. Hence, PYM is an EJC disassembly factor that acts both in vitro and in living cells, and that antagonizes important EJC functions.
The growth of daughter cells in budding yeast is a classic model for investigating mechanisms involved in asymmetric cell division. An unexpected collaboration between the DEAD-box protein Dbp5 and the nuclear transport receptor Kap104 controls localized protein synthesis at the bud tip during mitosis.
Nonsense-mediated mRNA decay (NMD) degrades mRNAs carrying premature translation termination codons (PTCs). Although the core process and several NMD effectors are conserved among species, the involvement of a splicing-dependent signal seems to be specific for mammalian PTC definition. Still, recent data shed new light on physical parameters and mechanistic pathways involved in NMD. Here, we examine these findings, updating the roles for potential NMD players, such as the exon junction complex and the cytoplasmic poly(A)-binding protein 1 - the former acting as enhancer rather than an essential factor and the latter functioning as NMD repressor.
The rates of processing and export of a variety of nuclear RNA species into the cytoplasmic compartment were studied by determining the rates of incorporation of tritiated uridine into nuclear and cytoplasmic RNA species. In exponentially growing cells, the rates of nuclear processing/export varied by more than a factor of ten for the six different mRNA species that were examined. Differences in the rates did not appear to be correlated with either the number or the sizes of introns in the genes for the RNA species. When cells were maintained under conditions of reduced protein synthesis (starvation for isoleucine and glutamine or exposure to cycloheximide), the processing rates for each species decreased by a factor of about 3. The decrease was not caused by the inability of hnRNA to associate with proteins, since the nuclear RNP distribution appeared normal in amino acid-starved cells.
We have analyzed the contributions to cytoplasmic stability in an mRNA species with a very short half-life (human c-fos) and an mRNA species with a very long half-life (human beta-globin). When the human c-fos promoter was used to drive the expression of human c-fos, beta-globin, and chimeric DNAs between c-fos and beta-globin in transfected cells, a pulse of mRNA synthesis was obtained following induction of transcription by refeeding quiescent cells with medium containing 15% calf serum. The mRNA half-life was determined by using Northern (RNA) blot analysis of mRNAs prepared at various times following the pulse of transcription. Under these conditions human c-fos mRNA exhibited a half-life of 6.6 min and human beta-globin mRNA exhibited a half-life of 17.5 h. Replacement of the 3' end of the c-fos mRNA with the 3' end of the beta-globin mRNA increased the half-life of the resultant RNA from 6.6 to 34 min. The reciprocal chimera had a half-life of 34.6 min compared with the 17.5-h half-life of beta-globin mRNA. These results suggest that sequences which make a major contribution to mRNA stability reside in the 3' end of either or both molecules. A chimera in which the 5' untranslated region of globin was replaced by part of the 5' untranslated region of fos led to destabilization of the encoded mRNA. This construct produced an mRNA with a half-life of 6.8 h instead of the 17.5-h half-life of globin. This result suggests that additional determinants of stability reside in the 5' end of these mRNA molecules. Substitution of part of the 5' untranslated region of fos by the 5' untranslated region of beta-globin yielded an mRNA with stability similar to fos mRNA. These results suggest that interactions among sequences within each mRNA contribute to the stability of the respective molecules.
The guanine nucleotide dissociation and GTPase reactions of Ran, a Ras-related nuclear protein, have been investigated using different fluorescence techniques to determine how these reactions are stimulated by the guanine nucleotide exchange factor RCC1 and the other regulatory protein, RanGAP1 (GTPase-activating protein). The intrinsic GTPase of Ran is one-tenth of the rate of p21ras and is even lower in the Ran(Q69L) mutant. Under saturating conditions the rate constant for the RanGAP1 stimulated GTPase reaction is 2.1 s-1 at 25 degrees C, which is a 10(5)-fold stimulation, whereas RanGAP1 has no effect on Ran(Q69L). The intrinsic guanine nucleotide dissociation rates of Ran are also very low and are likewise increased 10(5)-fold by the exchange factor RCC1. Methods to describe the reaction kinetically are presented. The Ran(T24N) mutant, which is analogous to the S17N mutant of p21ras, has decreased relative affinities for both GDP/GTP and favors GDP binding. However, it was found to interact almost normally with RCC1. The combination of these properties leads to stabilization of the Ran(T24N)-RCC1 complex and may result in vivo in depletion of RCC1 available for stimulating guanine nucleotide exchange.
In addition to the m7G cap structure, the length of the 5' UTR and the position and context of the AUG initiator codon (which have been discussed elsewhere in this volume), higher order structures within mRNA represent a critical parameter for translation. The role of RNA structure in translation initiation will be considered primarily, although structural elements have also been found to affect translation elongation and termination. We will first describe the different effects of higher order RNA structures per se, and then consider specific examples of RNA structural elements which control translation initiation by providing binding sites for regulatory proteins.
A cap-binding protein complex (CBC) present in the nuclei of HeLa cells has been characterized. Purified CBC consists of two previously identified proteins, CBP80 and CBP20. These proteins are shown to cofractionate to apparent homogeneity and to be coimmunoprecipitable with anti-CBP80 antibodies. Analysis of the inhibition of pre-mRNA splicing in vitro and in vivo by chemically modified analogs of the cap structure, and of the binding of these analogs to CBC in vitro, suggests a role for the complex in splicing. Extracts immunodepleted of CBC do not efficiently splice an adenoviral pre-mRNA owing to blockage of an early step in splicing complex formation. CBC may therefore play a role in pre-mRNA recognition.
The nuclear Ras-related protein Ran binds guanine nucleotide and is involved in cell cycle regulation. Models of the signal pathway predict Ran to be active as Ran.GTP at the initiation of S phase upon activation by the nucleotide exchange factor RCC1 and to be inactivated for the onset of mitosis by hydrolysis of bound GTP. Here a nuclear homodimeric 65-kDa protein, RanGAP1, is described, which we believe to be the immediate antagonist of RCC1. It was purified from HeLa cell lysates and induces GTPase activity of Ran, but not Ras, by more than 3 orders of magnitude. The Ran mutant Q69L, modeled after RasQ61L, which is unable to hydrolyze bound GTP, is insensitive to RanGAP1.
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As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.
Importin-alpha mediates nuclear protein import by binding nuclear localization signals and importin-beta. We find approximately 30% of SRP1p, the yeast importin-alpha, in a nuclear complex with the Saccharomyces cerevisiae nuclear cap-binding protein complex (CBC). Similarly, a large fraction of Xenopus CBC is associated with importin-alpha in the nucleus. CBC promotes nuclear export of capped U snRNAs and shuttles between nucleus and cytoplasm. The CBC-importin-alpha complex binds specifically to capped RNA, suggesting that CBC might shuttle while bound to importin-alpha. Strikingly, importin-beta binding displaces the RNA from the CBC-importin-alpha complex. Thus, the commitment of CBC for nuclear reentry triggers the release of the export substrate into the cytoplasm. We provide evidence for a mechanism that ensures that importin-mediated RNA release is a specifically cytoplasmic event.
We have cloned and sequenced cDNA for human karyopherin beta2, also known as transportin. In a solution binding assay, recombinant beta2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1's previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin beta1, karyopherin beta2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both beta1 and beta2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, beta2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of beta2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of beta2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin beta2 inhibited karyopherin alpha/beta1-mediated import of a classical NLS containing substrate and, vice versa, beta1 inhibited beta2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of beta1 and beta2 to repeat nucleoporins.
Nonsense codons between position 14 within the first exon and position 193 within the penultimate exon of the human gene for triosephosphate isomerase reduce mRNA abundance to 25% of normal. The reduction in abundance is due to the decay of newly synthesized mRNA that copurifies with nuclei. TPI mRNA that copurifies with cytoplasm is immune to decay. We show here that immunity is not due to the failure of nonsense-containing mRNA to form polysomes. This finding indicates that cytoplasmic mRNA, in contrast to nucleus-associated mRNA, may have lost one or more factors that are required for nonsense-mediated decay or gained one or more factors that confer immunity to nonsense-mediated decay.
The X-ray structure of the eukaryotic translation initiation factor 4E (eIF4E), bound to 7-methyl-GDP, has been determined at 2.2 A resolution. eIF4E recognizes 5' 7-methyl-G(5')ppp(5')N mRNA caps during the rate-limiting initiation step of translation. The protein resembles a cupped hand and consists of a curved, 8-stranded antiparallel beta sheet, backed by three long alpha helices. 7-methyl-GDP binds in a narrow cap-binding slot on the molecule's concave surface, where 7-methyl-guanine recognition is mediated by base sandwiching between two conserved tryptophans, plus formation of three hydrogen bonds and a van der Waals contact between its N7-methyl group and a third conserved tryptophan. The convex dorsal surface of the molecule displays a phylogenetically conserved hydrophobic/acidic portion, which may interact with other translation initiation factors and regulatory proteins.
The cap structure that is characteristic of all polymerase-II-transcribed RNAs has been shown to play an important role in many aspects of RNA metabolism including RNA processing, RNA nuclear transport, and translation initiation. The effects of the cap structure on these different processes is mediated by proteins that recognise and bind to it, and are therefore generically called cap-binding proteins. For example, the cap-binding protein eIF4E, in a complex with other proteins, mediates the effect of the cap on the initiation of translation. EIF-4E is predominantly localised in the cytoplasm. In the last five years, it has been demonstrated that a second cap-binding protein complex, which is mainly localised in the nucleus, mediates the stimulatory effects of the cap in nuclear processes such as pre-mRNA splicing, RNA 3'-end formation, and RNA nuclear export. The purpose of this review is to summarise our current knowledge on the role of the cap structure and of the cap-binding protein complex in nuclear RNA metabolism and present evidence that at least some processes may be coupled in vivo.
eIF4E, the mRNA cap binding protein, is a master switch that controls eukaryotic translation. To be active, it must bind eIF4G and form the eIF4F complex, which also contains eIF4A. Translation is downregulated by association of eIF4E with 4E-BP, which occupies the eIF4G binding site. Signalling events acting on 4E-BP cause it to dissociate from eIF4E, and eIF4E is then free to bind eIF4G to form the active eIF4F complex. We have solved the structure of the yeast eIF4E/m7Gpp complex in a CHAPS micelle. We determined the position of the second nucleotide in a complex with m7GpppA, and identified the 4E-BP binding site. eIF4E has a curved eight-stranded antiparallel beta-sheet, decorated with three helices on the convex face and three smaller helices inserted in connecting loops. The m7G of the cap is intercalated into a stack of tryptophans in the concave face. The 4E-BP binding site is located in a region encompassing one edge of the beta-sheet, the adjacent helix a2 and several regions of non-regular secondary structure. It is adjacent to, but does not overlap the cap-binding site.
`Time, that takes survey of all the world, Must have a stop.'Shakespeare, Henry IV, Part One
Generally, mRNAs that prematurely terminate translation are abnormally low in abundance. In the case of mammalian cells, nonsense codons most often mediate a reduction in the abundance of newly synthesized, nucleus-associated mRNA by a mechanism that is not well understood. With the aim of defining cis-acting sequences that are important to the reduction process, the effects of particular beta-globin gene rearrangements on the metabolism of beta-globin mRNAs harboring one of a series of nonsense codons have been assessed. Results indicate that nonsense codons located 54 bp or more upstream of the 3'-most intron, intron 2, reduce the abundance of nucleus-associated mRNA to 10-15% of normal without altering the level of either of the two introns within pre-mRNA. The level of cytoplasmic mRNA is also reduced to 10-15% of normal, indicating that decay does not take place once the mRNA is released from an association with nuclei into the cytoplasm. A nonsense codon within exon 2 that does not reduce mRNA abundance can be converted to the type that does by (1) inserting a sufficiently large in-frame sequence immediately upstream of intron 2 or (2) deleting and reinserting intron 2 a sufficient distance downstream of its usual position. These findings indicate that only those nonsense codons located more than 54 bp upstream of the 3'-most intron reduce beta-globin mRNA abundance, which is remarkably consistent with which nonsense codons within the triosephosphate isomerase (TPI) gene reduce TPI mRNA abundance. We propose that the 3'-most exon-exon junction of beta-globin mRNA and, possibly, most mRNAs is marked by the removal of the 3'-most intron during pre-mRNA splicing and that the "mark" accompanies mRNA during transport to the cytoplasm. When cytoplasmic ribosomes terminate translation more than 54 nt upstream of the mark during or immediately after transport, the mRNA is subjected to nonsense-mediated decay. The finding that deletion of beta-globin intron 2 does not appreciably alter the effect of any nonsense codon on beta-globin mRNA abundance suggests that another cis-acting sequence functions in nonsense-mediated decay comparably to intron 2, at least in the absence of intron 2, possibly as a fail-safe mechanism. The analysis of deletions and insertions indicates that this sequence resides within the coding region and can be functionally substituted by intron 2.
The import of proteins into the nucleus is dependent on
cis
-acting targeting sequences, nuclear localization signals (NLSs), and members of the nuclear transport receptor (importin-β-like) superfamily. The most extensively characterized import pathway, often termed the classical pathway, is utilized by many basic-type (lysine-rich) NLSs and requires an additional component, importin α, to serve as a bridge between the NLS and the import receptor importin β. More recently, it has become clear that a variety of proteins enter the nucleus via alternative import receptors and that their NLSs bind directly to those receptors. By using the digitonin-permeabilized cell system for protein import in vitro, we have defined the import pathway for the Rex protein of human T-cell leukemia virus type 1. Interestingly, the arginine-rich NLS of Rex uses importin β for import but does so by a mechanism that is importin α independent. Based on the ability of the Rex NLS to inhibit the import of the lysine-rich NLS of T antigen and of both NLSs to be inhibited by the domain of importin α that binds importin β (the IBB domain), we infer that the Rex NLS interacts with importin β directly. In addition, and in keeping with other receptor-mediated nuclear import pathways, Rex import is dependent on the integrity of the Ran GTPase cycle. Based on these results, we suggest that importin β can mediate the nuclear import of arginine-rich NLSs directly, or lysine-rich NLSs through the action of importin α.
Serine/arginine-rich proteins (SR proteins) are mainly involved in the splicing of precursor mRNA. RS domains are also found in proteins that have influence on other aspects of gene expression. Proteins that contain an RS domain are often located in the speckled domains of the nucleus. Here we show that the RS domain derived from a human papillomavirus E2 transcriptional activator can target a heterologous protein to the nucleus, as it does in many other SR proteins, but insufficient for localization in speckles. By using E2 as a bait in a yeast two-hybrid screen, we identified a human importin-beta family protein that is homologous to yeast Mtr10p and almost identical to human transportin-SR. This transportin-SR2 (TRN-SR2) protein can interact with several cellular SR proteins. More importantly, we demonstrated that TRN-SR2 can directly interact with phosphorylated, but not unphosphorylated, RS domains. Finally, an indirect immunofluoresence study revealed that a transiently expressed TRN-SR2 mutant lacking the N-terminal region becomes localized to the nucleus in a speckled pattern that coincides with the distribution of the SR protein SC35. Thus, our results likely reflect a role of TRN-SR2 in the cellular trafficking of phosphorylated SR proteins.