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ABSTRACT: Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that in mammals generally occurs upon recognition of a premature termination codon (PTC) during a pioneer round of translation. This round involves newly synthesized mRNA that is bound at its 5' end by the cap-binding protein (CBP) heterodimer CBP80-CBP20. Here we show that precluding the binding of the NMD factor UPF1 to CBP80 inhibits NMD at two steps: the association of SMG1 and UPF1 with the two eukaryotic release factors (eRFs) during SURF complex formation at a PTC, and the subsequent association of SMG1 and UPF1 with an exon-junction complex. We also demonstrate that UPF1 binds PTC-containing mRNA more efficiently than the corresponding PTC-free mRNA in a way that is promoted by the UPF1-CBP80 interaction. A unifying model proposes a choreographed series of protein-protein interactions occurring on an NMD target.
Molecular cell 08/2010; 39(3):396-409. · 14.61 Impact Factor
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ABSTRACT: Factors affecting translation of mRNA contribute to the complexity of eukaryotic proteomes. In some cases, translation of a particular mRNA can generate multiple proteins. However, the factors that determine whether ribosomes initiate translation from the first AUG codon in the transcript, from a downstream codon, or from multiple sites are not completely understood. Various mRNA properties, including AUG codon-accessibility and 5' leader length have been proposed as potential determinants that affect where ribosomes initiate translation. To explore this issue, we performed studies using synthetic mRNAs with two in-frame AUG codons-both in excellent context. Open reading frames initiating at AUG1 and AUG2 encode large and small isoforms of a reporter protein, respectively. Translation of such an mRNA in COS-7 cells was shown to be 5' cap-dependent and to occur efficiently from both AUG codons. AUG codon-accessibility was modified by using two different elements: an antisense locked nucleic acid oligonucleotide and an exon-junction complex. When either element was used to mask AUG1, the ratio of the proteins synthesized changed, favoring the smaller (AUG2-initiated) protein. In addition, we observed that increased leader length by itself changed the ratio of the proteins and favored initiation at AUG1. These observations demonstrate that initiation codon selection is affected by various factors, including AUG codon-accessibility and 5' leader length, and is not necessarily determined by the order of AUG codons (5'→3'). The modulation of AUG codon accessibility may provide a powerful means of translation regulation in eukaryotic cells.
PLoS ONE 01/2010; 5(11):e15057. · 4.09 Impact Factor
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ABSTRACT: We demonstrate the presence of a functional internal ribosome entry site (IRES) within the 5' leader (designated 5L) from a variant of bicistronic mRNAs that encode the pp14 and RLORF9 proteins from Marek's disease virus (MDV) serotype 1. Transcribed as a 1.8-kb family of immediate-early genes, the mature bicistronic mRNAs have variable 5' leader sequences due to alternative splicing or promoter usage. Consequently, the presence or absence of the 5L IRES in the mRNA dictates the mode of pp14 translation and leads to the production of two pp14 isoforms that differ in their N-terminal sequences. Real-time reverse transcription-quantitative PCR indicates that the mRNA variants with the 5L IRES is two to three times more abundant in MDV-infected and transformed cells than the mRNA variants lacking the 5L IRES. A common feature to all members of the 1.8-kb family of transcripts is the presence of an intercistronic IRES that we have previously shown to control the translation of the second open reading frame (i.e., RLORF9). Investigation of the two IRESs residing in the same bicistronic reporter mRNA revealed functional synergism for translation efficiency. In analogy with allosteric models in proteins, we propose IRES allostery to describe such a novel phenomenon. The functional implications of our findings are discussed in relation to host-virus interactions and translational control.
Journal of Virology 09/2009; 83(24):12769-78. · 5.40 Impact Factor
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ABSTRACT: Nonsense-mediated decay (NMD) in eukaryotic cells largely functions as a quality control mechanism by degrading faulty mRNAs that terminate translation prematurely. In recent years it has become evident that NMD also eliminates a subset of naturally occurring mRNA during proper gene expression. The mechanism of NMD in mammalian cells can be distinguished from the mechanism in, for example, Saccharomyces cerevisiae or Caenorhabditis elegans, by its apparent restriction to newly synthesized mRNA during a pioneer round of translation. This dependence can be explained by the need for at least one exon-exon junction complex (EJC) that is deposited on newly synthesized mRNA during the process of pre-mRNA splicing. Additionally, mammalian-cell NMD is promoted by the cap-binding protein heterodimer CBP80/20 that also typifies newly synthesized mRNA. When translation terminates sufficiently upstream of an EJC, the NMD factor Up-frameshift (Upf)1 is thought to join the stable EJC constituent NMD factors Upf2 and Upf3 or Upf3X (also called Upf3a or Upf3b, respectively), and undergo phosphorylation. Phosphorylation appears to trigger translational repression and mRNA decay. Although there are established rules for what generally defines an NMD target in mammalian cells, as with any rule there are exceptions and, thus, the need to experimentally verify individual mRNAs as bona fide targets of NMD. This chapter provides guidelines and protocols for how to define NMD targets using cultured mammalian cells.
Methods in enzymology 02/2008; 449:177-201. · 1.90 Impact Factor
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ABSTRACT: Nonsense-mediated mRNA decay (NMD) generally eliminates messenger RNAs that prematurely terminate translation and occurs in all eukaryotes that have been studied, although with mechanistic variations. In mammals, NMD seems to be restricted to newly synthesized mRNA that is bound by the cap-binding heterodimer CBP80-CBP20 (CBP80/20) and typically has at least one exon junction complex (EJC) situated downstream of the nonsense codon and added post-splicing. However, mammalian NMD can also target spliced mRNA lacking an EJC downstream of the nonsense codon. Here we provide evidence that this additional pathway, known as failsafe NMD, likewise seems to be restricted to CBP80/20-bound mRNA and does not detectably target its subsequently remodeled product, eIF4E-bound mRNA. Our studies, including analyses of factor dependence, reveal important shared features of the two mammalian-cell NMD pathways as well as fundamental differences between NMD in mammals and Saccharomyces cerevisiae.
Nature Structural & Molecular Biology 11/2007; 14(10):974-9. · 12.71 Impact Factor
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ABSTRACT: Turnip yellow mosaic virus (TYMV) RNA directs the translation of two overlapping open reading frames. Competing models have been previously published to explain ribosome access to the downstream polyprotein cistron. The Trojan horse model, based on cell-free experiments, proposes noncanonical cap-independent initiation in which the 3'-terminal tRNA-like structure (TLS) functionally replaces initiator tRNA, and the valine bound to the TLS becomes cis-incorporated into viral protein. The initiation coupling model, based on in vivo expression and ribosome toe-printing studies, proposes a variation of canonical leaky scanning. Here, we have re-examined the wheat germ extract experiments that led to the Trojan horse model, incorporating a variety of controls. We report that (1) translation in vitro from the polyprotein AUG of TYMV RNA is unchanged after removal of the 3' TLS but is stimulated by the presence of a 5'-cap; (2) the presence of free cap analog or edeine (which interferes with initiation at the ribosomal P site and its tRNA(i) (Met) involvement) inhibits translation from the polyprotein AUG; (3) the toe-prints of immediately post-initiation ribosomes on TYMV RNA are similar with and without an intact TLS; and (4) significant deacylation of valyl-TYMV RNA in wheat germ extract can complicate the detection of cis-incorporation. These results favor the initiation coupling model.
RNA 02/2007; 13(1):129-37. · 5.09 Impact Factor
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ABSTRACT: TYMV RNA supports the translation of two proteins, p69 and p206, from AUG initiation codons 7 nucleotides apart. We have studied the translation of this overlapping dicistronic mRNA with luciferase reporter RNAs electroporated into cowpea protoplasts and in toe-printing studies that map ribosomes stalled during initiation in wheat germ extracts. Agreement between these two assays indicates that the observed effects reflect ribosome initiation events. The robust expression from the downstream AUG206 codon was dependent on its closeness to the upstream AUG69 codon. Stepwise separation of these codons resulted in a gradual increase in upstream initiation and decrease in downstream initiation, and expression was converted from dicistronic to monocistronic. Selection by ribosomes for initiation between the nearby AUG codons was responsive to the sequence contexts that govern leaky scanning, but the normally strong position effect favoring upstream initiation was greatly diminished. Similar dicistronic expression was supported for RNAs with altered initiation sequences and for RNAs devoid of flanking viral sequences. Closely spaced AUG codons may thus represent an under-recognized strategy for bicistronic expression from eukaryotic mRNAs. The initiation behavior observed in these studies suggests that 5'-3' ribosome scanning involves backward excursions averaging about 15 nucleotides.
RNA 08/2006; 12(7):1338-49. · 5.09 Impact Factor
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ABSTRACT: This unit describes the analysis of the translation-regulating properties of viral RNAs in cell-based assays using a luciferase reporter system. Electroporation and polyethylene glycol-mediated introduction of luciferase reporter RNA into cowpea protoplasts are also presented. The Commentary section discusses employing the luciferase reporter system to study translational control by the 5'- and 3'-untranslated regions of a viral mRNA. Inoculum RNA quality and half-life after delivery to the protoplasts should be monitored closely for careful investigation of translational regulation elements.
Current protocols in microbiology 11/2005; Chapter 16:Unit 16K.2.
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ABSTRACT: The translation efficiency of an mRNA molecule is typically determined by its 5'- and/or 3'-untranslated regions (UTRs). Previously, we have found that the 3'-UTR of Turnip yellow mosaic virus (TYMV) RNA enhances translation synergistically with a 5' cap. Here, we use a luciferase reporter system in cowpea protoplasts to show that the 5' 217 nucleotides from TYMV genomic RNA enhance expression relative to a vector-derived 17-nucleotide 5'-UTR. Maximum expression was observed from RNAs with a cap and both 5' and 3' TYMV sequences. In paired reporter constructs, the 5' 217 nucleotides harboring the UTR and the first 43 or 41 codons of the two overlapping TYMV open reading frames (ORFs), ORF-69 and ORF-206, respectively, were fused in frame with the luciferase gene. This allowed expression from the initiation codon of each ORF (AUG69 and AUG206) to be monitored separately but from the normal sequence environment. Expression from both AUG codons was heavily dependent on a 5' cap, with a threefold-higher expression occurring from AUG69 than from AUG206 in the presence of the genomic 3'-UTR. Changes that interrupted the cap/3'-UTR synergy (i.e., removal of the cap or TYMV 3'-UTR) resulted in a higher proportion of initiation from AUG206. Mutation of the 3'-UTR to prevent aminoacylation, as well as deletion of 75% of the 5'-UTR, likewise resulted in a lower ratio of expression from AUG69 relative to AUG206. Mutation of each AUG initiation codon increased initiation from the other. Taken together, these results do not fully conform to the expectations of standard leaky ribosomal scanning and leave open the precise mechanism of ribosome commitment to AUG69 and AUG206. However, our observations do not support a recent proposal based on in vitro studies in which the 3'-UTR is proposed to direct cap-independent initiation specifically at AUG206 and not at AUG69 (S. Barends et al., Cell 112:123-129, 2003).
Journal of Virology 10/2004; 78(17):9325-35. · 5.40 Impact Factor
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ABSTRACT: The genomic RNA of Turnip yellow mosaic virus (TYMV) has an 82-nucleotide-long tRNA-like structure at its 3'-end that can be valylated and then form a stable complex with translation elongation factor eEF1A.GTP. Transcription of this RNA by TYMV RNA-dependent RNA polymerase (RdRp) to yield minus strands has previously been shown to initiate within the 3'-CCA sequence. We have now demonstrated that minus strand synthesis is strongly repressed upon the binding of eEF1A.GTP to the valylated viral RNA. eEF1A.GTP had no effect on RNA synthesis templated by non-aminoacylated RNA. Higher eEF1A.GTP levels were needed to repress minus strand synthesis templated by valyl-EMV TLS RNA, which binds eEF1A.GTP with lower affinity than does valyl-TYMV RNA. Repression by eEF1A.GTP was also observed with a methionylated variant of TYMV RNA and with aminoacylated tRNAHis, tRNAAla, and tRNAPhe transcripts. It is proposed that minus strand repression by eEF1A.GTP binding occurs early during infection to help coordinate the competing translation and replication functions of the genomic RNA.
Virology 04/2004; 321(1):47-56. · 3.35 Impact Factor
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ABSTRACT: Many positive stand RNA viral genomes lack the poly(A) tail that is characteristic of cellular mRNAs and that promotes translation in cis. The 3' untranslated regions (UTRs) of such genomes are expected to provide similar translation-enhancing properties as a poly(A) tail, yet the great variety of 3' sequences suggests that this is accomplished in a range of ways. We have identified a translational enhancer present in the 3' UTR of Turnip yellow mosaic virus (TYMV) RNA using luciferase reporter RNAs with generic 5' sequences transfected into plant cells. The 3' terminal 109 nucleotides comprising the tRNA-like structure (TLS) and an upstream pseudoknot (UPSK) act in synergy with a 5'-cap to enhance translation, with a minor contribution in stabilizing the RNA. Maximum enhancement requires that the RNA be capable of aminoacylation, but either the native valine or engineered methionine is acceptable. Mutations that decrease the affinity for translation elongation factor eEF1A (but also diminish aminoacylation efficiency) strongly decrease translational enhancement, suggesting that eEF1A is mechanistically involved. The UPSK seems to act as an important, though nonspecific, spacer element ensuring proper presentation of a functional TLS. Our studies have uncovered a novel type of translational enhancer and a new role for a plant viral TLS.
Virology 04/2004; 321(1):36-46. · 3.35 Impact Factor
