[Show abstract][Hide abstract] ABSTRACT: Internal ribosome entry site (IRES) elements are high order RNA structures that promote internal initiation of translation allowing protein synthesis under situations that compromise the general cap-dependent translation mechanism. Picornavirus IRES are highly efficient elements with a modular RNA structure organization. Here we investigated the impact of Mg(2+) concentration on the local flexibility and solvent accessibility of the foot-and-mouth disease virus (FMDV) IRES measured by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) reactivity and hydroxyl radical cleavage. We have found that Mg(2+) concentration affects the organization of discrete IRES regions, mainly the apical region of domain 3, the 10-nt loop of domain 4 and the pyrimidine tract of domain 5. In support of the impact of RNA structure for IRES activity, substitution or deletion mutants of the 10-nt loop of domain 4 impair internal initiation. In addition, divalent cations affect the binding of eIF4G, an initiation factor essential for IRES-dependent translation that interacts with domain 4. Binding of eIF4G is favored by the local RNA flexibility adopted at low Mg(2+) concentration while eIF4B interacts with the IRES independently of the compactness of the RNA structure. Our study shows that the IRES element adopts a near native structure in the absence of proteins, shedding light on the influence of Mg(2+) ions on the local flexibility and binding of eIF4G in a model IRES element. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Maize heat shock protein of 101 KDa (HSP101) is essential for thermotolerance induction in this plant. The mRNA encoding this protein harbors an IRES element in the 5'UTR that mediates cap-independent translation initiation. In the current work it is demonstrated that hsp101 IRES comprises the entire 5'UTR sequence (150 nts), since deletion of 17 nucleotides from the 5' end decreased translation efficiency by 87% compared to the control sequence. RNA structure analysis of maize hsp101 IRES revealed the presence of three stem-loops toward its 5' end, whereas the remainder sequence contains a great proportion of unpaired nucleotides. Furthermore, HSP90 protein was identified by mass spectrometry as the protein preferentially associated with the maize hsp101 IRES. In addition, it has been found that eIFiso4G rather than eIF4G initiation factor mediates translation of the maize hsp101 mRNA.
PLoS ONE 01/2014; 9(9):e107459. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: More than half of the human genome is made of transposable elements whose ongoing mobilization is a driving force in genetic diversity; however, little is known about how the host regulates their activity. Here, we show that the Microprocessor (Drosha-DGCR8), which is required for microRNA biogenesis, also recognizes and binds RNAs derived from human long interspersed element 1 (LINE-1), Alu and SVA retrotransposons. Expression analyses demonstrate that cells lacking a functional Microprocessor accumulate LINE-1 mRNA and encoded proteins. Furthermore, we show that structured regions of the LINE-1 mRNA can be cleaved in vitro by Drosha. Additionally, we used a cell culture-based assay to show that the Microprocessor negatively regulates LINE-1 and Alu retrotransposition in vivo. Altogether, these data reveal a new role for the Microprocessor as a post-transcriptional repressor of mammalian retrotransposons and a defender of human genome integrity.
[Show abstract][Hide abstract] ABSTRACT: Picornavirus RNAs initiate translation using a 5' end-independent mechanism based on internal ribosome entry site (IRES) elements. Despite performing similar functions, IRES elements present in genetically distant RNAs differ in primary sequence, RNA secondary structure and trans-acting factors requirement. The lack of conserved features amongst IRESs represents obstacles for the understanding of the internal initiation process. However, RNA structure is tightly linked topicornavirus IRES activity, consistent with the conservation of RNA motifs. This study extends the functional relevance of evolutionary conserved motifs of foot-and-mouth disease virus (FMDV) IRES. SHAPE structural analysis of mutant IRESs revealed local changes in RNA flexibility indicating the existence of an interactive structure constrained by lateral bulges that maintain the RNA conformation necessary for IRES-mediated translation.
[Show abstract][Hide abstract] ABSTRACT: Gene expression control largely depends on ribonucleoprotein complexes regulating mRNA translation. Initiation of translation in mRNAs that overcome cap-dependent translation inhibition is often driven by internal ribosome entry site (IRES) elements, whose activity is regulated by multifunctional RNA-binding factors. Here we show that Gemin5 interacts preferentially with a specific domain of a viral IRES consisting of a hairpin flanked by A/U/C-rich sequences. RNA-binding assays using purified proteins revealed that Gemin5-IRES interaction depends on the C-terminal region of the protein. Consistent with this novel finding, the C-terminal region of Gemin5, but not the N-terminal region, impaired translation. Furthermore, RNA selective 2'hydroxyl acylation analysed by primer extension (SHAPE) reactivity demonstrated that addition of purified Gemin5 to IRES mRNA induced the specific protection of residues around the hairpin of the IRES element. We further demonstrate that Gemin5 out-competed SHAPE reactivity variations induced by the IRES-binding factor PTB, leading to a local conformational change in the IRES structure. Together, our data unveil the inhibitory mechanism of Gemin5 on IRES-mediated translation.
Nucleic Acids Research 12/2012; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The composition of the cellular proteome is under the control of multiple processes, one of the most important being translation initiation. The majority of eukaryotic cellular mRNAs initiates translation by the cap-dependent or scanning mode of translation initiation, a mechanism that depends on the recognition of the m(7)G(5')ppp(5')N, known as the cap. However, mRNAs encoding proteins required for cell survival under stress bypass conditions inhibitory to cap-dependent translation; these mRNAs often harbor internal ribosome entry site (IRES) elements in their 5'UTRs that mediate internal initiation of translation. This mechanism is also exploited by mRNAs expressed from the genome of viruses infecting eukaryotic cells. In this paper we discuss recent advances in understanding alternative ways to initiate translation across eukaryotic organisms.
Comparative and Functional Genomics 01/2012; 2012:391546. · 0.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RNA structure plays a fundamental role in internal initiation of translation. Picornavirus internal ribosome entry site (IRES) are long, efficient cis-acting elements that recruit the ribosome to internal mRNA sites. However, little is known about long-range constraints determining the IRES RNA structure. Here, we sought to investigate the functional and structural relevance of the invariant apical stem of a picornavirus IRES. Mutation of this apical stem revealed better performance of G:C compared with C:G base pairs, demonstrating that the secondary structure solely is not sufficient for IRES function. In turn, mutations designed to disrupt the stem abolished IRES activity. Lack of tolerance to accept genetic variability in the apical stem was supported by the presence of coupled covariations within the adjacent stem-loops. SHAPE structural analysis, gel mobility-shift and microarrays-based RNA accessibility revealed that the apical stem contributes to maintain IRES RNA structure through the generation of distant interactions between two adjacent stem-loops. Our results demonstrate that a highly interactive structure constrained by distant interactions involving invariant G:C base pairs plays a key role in maintaining the RNA conformation necessary for IRES-mediated translation.
Nucleic Acids Research 07/2011; 39(19):8572-85. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Picornavirus RNA translation is driven by the internal ribosome entry site (IRES) element. The impact of RNA structure on the foot-and-mouth disease virus (FMDV) IRES activity has been analyzed using Selective 2'Hydroxyl Acylation analyzed by Primer Extension (SHAPE) and high throughput analysis of RNA conformation by antisense oligonucleotides printed on microarrays. SHAPE reactivity revealed the self-folding capacity of domain 3 and evidenced a change of RNA structure in a defective GNRA mutant. A modified RNA conformation of this mutant was also evidenced by RNA accessibility to oligonucleotides. Interestingly, comparison of nucleotide reactivity with RNA accessibility revealed that SHAPE reactive nucleotides corresponding to the GNRA motif were not accessible to their respective target oligonucleotides. The differential response was observed both in domain 3 and the entire IRES. Our results demonstrate distant effects of the GNRA motif in the domain 3 RNA conformation, and highlight the modular organization of a picornavirus IRES.
[Show abstract][Hide abstract] ABSTRACT: Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. We have previously shown that a structural motif within the foot-and-mouth-disease virus IRES is recognized in vitro as substrate for the Synechocystis sp. RNase P ribozyme. Here we show that this structure-dependent endonuclease recognizes the IRES element in cultured cells, leading to inhibition of translation. Inhibition of IRES activity was dependent on the expression of the active ribozyme RNA subunit. Moreover, expression of the antisense sequence of the ribozyme did not inhibit IRES activity, demonstrating that stable RNA structures located upstream of the IRES element do not interfere with internal initiation. RNAs carrying defective IRES mutants that were substrates of the ribozyme in vivo revealed an increased translation of the reporter in response to the expression of the active ribozyme. In support of RNA cleavage, subsequent analysis of the translation initiation manner indicated a switch from IRES-dependent to 5'-end-dependent translation of RNase P target RNAs. We conclude that the IRES element is inactivated by expression in cis of RNase P in the cytoplasm of cultured cells, providing a promising antiviral tool to combat picornavirus infections. Furthermore, our results reinforce the essential role of the structural motif that serves as RNase P recognition motif for IRES activity.
[Show abstract][Hide abstract] ABSTRACT: In eukaryotic cells translation initiation occurs through two alternative mechanisms, a cap-dependent operating in the majority of mRNAs, and a 5'-end-independent driven by internal ribosome entry site (IRES) elements, specific for a subset of mRNAs. IRES elements recruit the translation machinery to an internal position in the mRNA through a mechanism involving the IRES structure and several trans-acting factors. Here, we identified Gemin5 protein bound to the foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) IRES using two independent approaches, riboproteomic analysis and immunoprecipitation of photocrosslinked factors. Functional analysis performed in Gemin5 shRNA-depleted cells, or in in vitro translation reactions, revealed an unanticipated role of Gemin5 in translation control as a down-regulator of cap-dependent and IRES-driven translation initiation. Consistent with this, pull-down assays showed that Gemin5 forms part of two distinct complexes, a specific IRES-ribonucleoprotein complex and an IRES-independent protein complex containing eIF4E. Thus, beyond its role in snRNPs biogenesis, Gemin5 also functions as a modulator of translation activity.
Nucleic Acids Research 01/2009; 37(2):582-90. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A distinctive feature of positive-strand RNA viruses is the presence of high-order structural elements at the untranslated regions (UTR) of the genome that are essential for viral RNA replication. The RNA of all members of the family Picornaviridae initiate translation internally, via an internal ribosome entry site (IRES) element present in the 5' UTR. IRES elements consist of cis-acting RNA structures that usually require specific RNA-binding proteins for translational machinery recruitment. This specialized mechanism of translation initiation is shared with other viral RNAs, e.g. from hepatitis C virus and pestivirus, and represents an alternative to the cap-dependent mechanism. In cells infected with many picornaviruses, proteolysis or changes in phosphorylation of key host factors induces shut off of cellular protein synthesis. This event occurs simultaneously with the synthesis of viral gene products since IRES activity is resistant to the modifications of the host factors. Viral gene expression and RNA replication in positive-strand viruses is further stimulated by viral RNA circularization, involving direct RNA-RNA contacts between the 5' and 3' ends as well as RNA-binding protein bridges. In this review, we discuss novel insights into the mechanisms that control picornavirus gene expression and compare them to those operating in other positive-strand RNA viruses.
Journal of General Virology 04/2008; 89(Pt 3):611-26. · 3.13 Impact Factor