Upf proteins: highly conserved factors involved in nonsense mRNA mediated decay

Over 10% of genetic diseases are caused by mutations that introduce a premature termination codon in protein-coding mRNA. Nonsense-mediated mRNA decay (NMD) is an essential cellular pathway that degrades these mRNAs to prevent the accumulation of harmful partial protein products. NMD machinery is also increasingly appreciated to play a role in other essential cellular functions, including telomere homeostasis and the regulation of normal mRNA turnover, and is misregulated in numerous cancers. Hence, understanding and designing therapeutics targeting NMD is an important goal in biomedical science. The central regulator of NMD, the Upf1 protein, interacts with translation termination factors and contextual factors to initiate NMD specifically on mRNAs containing PTCs. The molecular details of how these contextual factors affect Upf1 function remain poorly understood. Here, we review plausible models for the NMD pathway and the evidence for the variety of roles NMD machinery may play in different cellular processes.     

An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3'-->5' degradation

Eukaryotic mRNAs containing premature termination codons are subjected to accelerated turnover, known as nonsense-mediated decay (NMD). Recognition of translation termination events as premature requires a surveillance complex, which includes the RNA helicase Upf1p. In Saccharomyces cerevisiae, NMD provokes rapid decapping followed by 5'-->3' exonucleolytic decay. Here we report an alternative, decapping-independent NMD pathway involving deadenylation and subsequent 3'-->5' exonucleolytic decay. Accelerated turnover via this pathway required Upf1p and was blocked by the translation inhibitor cycloheximide. Degradation of the deadenylated mRNA required the Rrp4p and Ski7p components of the cytoplasmic exosome complex, as well as the putative RNA helicase Ski2p. We conclude that recognition of NMD substrates by the Upf surveillance complex can target mRNAs to rapid deadenylation and exosome-mediated degradation.     

Nonsense-mediated mRNA decay in yeast does not require PAB1 or a poly(A) tail

Eukaryotic mRNAs harboring premature translation termination codons are recognized and rapidly degraded by the nonsense-mediated mRNA decay (NMD) pathway. The mechanism for discriminating between mRNAs that terminate translation prematurely and those subject to termination at natural stop codons remains unclear. Studies in multiple organisms indicate that proximity of the termination codon to the 3' poly(A) tail and the poly(A) RNA-binding protein, PAB1, constitute the critical determinant in NMD substrate recognition. We demonstrate that mRNA in yeast lacking a poly(A) tail can be destabilized by introduction of a premature termination codon and, importantly, that this mRNA is a substrate of the NMD machinery. We further show that, in cells lacking Pab1p, mRNA substrate recognition and destabilization by NMD are intact. These results establish that neither the poly(A) tail nor PAB1 is required in yeast for discrimination of nonsense-codon-containing mRNA from normal by NMD.     

The influence of UPF genes on the severity of SUP45 mutations

Eukaryotic cells possess special mechanism of the degradation of mRNAs containing premature termination codons (PTCs)--nonsense-mediated mRNA decay (NMD) pathway. In yeast Saccharomyces cerevisiae, the activity of this pathway depends on the recognition of the PTC by the translational machinery and interaction of translation termination factors eRF1 and eRF3 with Upf1, Upf2 and Upf3 proteins. Previously we have shown that decreasing of eRF1 amount causes an impairment of NMD. Here we show that deletion of either UPF1 or UPF2 increased viability of sup45 mutants, while effect of UPF3 deletion is allele-specific. Two-hybrid data have shown that aa 1-555 of eRF1 participate in interaction with Upf1. Deletion of each UPF gene leads to allosuppresson of ade1-14 mutation without changing eRF1 amount. Depletion of Upf1 does not influence synthetic lethality of sup45 and prion [PSI+]. It is possible that the absence of Upf1 (or its activator Upf2) leads to more effective formation of the translation termination complex and, consequently, increased viability of cells containing mutant termination factors.     

A new function for nonsense-mediated mRNA-decay factors

mRNAs often contain premature-termination (nonsense) codons as a result of mutations and RNA splicing errors. These nonsense codons cause rapid decay of the mRNAs that contain them, a phenomenon called nonsense-mediated mRNA decay (NMD). This response is thought to be a quality-control mechanism that protects cells from truncated dominant-negative proteins. Surprisingly, recent evidence strongly suggests that the NMD factors UPF1, UPF2, UPF3B, RNPS1, Y14 and MAGOH also promote translation of normal mRNAs in mammalian cells. This, along with an earlier discovery that NMD factors appear to dictate efficient translation termination, suggests that NMD factors do not merely function in RNA surveillance. These findings lead to the interesting question of why NMD factors evolved; are they for RNA-quality control or to promote efficient translation initiation and termination?     

Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae

Eukaryotic release factor 3 (eRF3) is implicated in translation termination and also interacts with the poly(A)-binding protein (PABP, Pab1 in yeast), a major player in mRNA metabolism. Despite conservation of this interaction, its precise function remains elusive. First, we showed experimentally that yeast eRF3 does not contain any obvious consensus PAM2 (PABP-interacting motif 2). Thus, in yeast this association is different from the well described interaction between the metazoan factors. To gain insight into the exact function of this interaction, we then analyzed the phenotypes resulting from deleting the respective binding domains. Deletion of the Pab1 interaction domain on eRF3 did not affect general mRNA stability or nonsense-mediated mRNA decay (NMD) pathway and induced a decrease in translational readthrough. Furthermore, combined deletions of the respective interacting domains on eRF3 and on Pab1 were viable, did not affect Pab1 function in mRNA stability and harbored an antisuppression phenotype. Our results show that in Saccharomyces cerevisiae the role of the Pab1 C-terminal domain in mRNA stability is independent of eRF3 and the association of these two factors negatively regulates translation termination.     

Inactivation of NMD increases viability of <Emphasis Type="Italic">sup45</Emphasis> nonsense mutants in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3.     

Infrequent translation of a nonsense codon is sufficient to decrease mRNA level

In many organisms nonsense mutations decrease the level of mRNA. In the case of mammalian cells, it is still controversial whether translation is required for this nonsense-mediated RNA decrease (NMD). Although previous analyzes have shown that conditions that impede translation termination at nonsense codons also prevent NMD, the residual level of termination was unknown in these experiments. Moreover, the conditions used to impede termination might also have interfered with NMD in other ways. Because of these uncertainties, we have tested the effects of limiting translation of a nonsense codon in a different way, using two mutations in the immunoglobulin mu heavy chain gene. For this purpose we exploited an exceptional nonsense mutation at codon 3, which efficiently terminates translation but nonetheless maintains a high level of mu mRNA. We have shown 1) that translation of Ter462 in the double mutant occurs at only approximately 4% the normal frequency, and 2) that Ter462 in cis with Ter3 can induce NMD. That is, translation of Ter462 at this low (4%) frequency is sufficient to induce NMD.     

Nonsense mutations in close proximity to the initiation codon fail to trigger full nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs containing premature translation termination codons. In mammalian cells, a termination codon is ordinarily recognized as "premature" if it is located greater than 50-54 nucleotides 5' to the final exon-exon junction. We have described a set of naturally occurring human beta-globin gene mutations that apparently contradict this rule. The corresponding beta-thalassemia genes contain nonsense mutations within exon 1, and yet their encoded mRNAs accumulate to levels approaching wild-type beta-globin (beta(WT)) mRNA. In the present report we demonstrate that the stabilities of these mRNAs with nonsense mutations in exon 1 are intermediate between beta(WT) mRNA and beta-globin mRNA carrying a prototype NMD-sensitive mutation in exon 2 (codon 39 nonsense; beta 39). Functional analyses of these mRNAs with 5'-proximal nonsense mutations demonstrate that their relative resistance to NMD does not reflect abnormal RNA splicing or translation re-initiation and is independent of promoter identity and erythroid specificity. Instead, the proximity of the nonsense codon to the translation initiation AUG constitutes a major determinant of NMD. Positioning a termination mutation at the 5' terminus of the coding region blunts mRNA destabilization, and this effect is dominant to the "50-54 nt boundary rule." These observations impact on current models of NMD.