Interaction of PABPC1 with the translation initiation complex is critical to the NMD resistance of AUG-proximal nonsense mutations

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'-5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.     

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 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.     

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.     

Leaky termination at premature stop codons antagonizes nonsense-mediated mRNA decay in S. cerevisiae

The Nonsense-Mediated mRNA Decay (NMD) pathway mediates the rapid degradation of mRNAs that contain premature stop mutations in eukaryotic organisms. It was recently shown that mutations in three yeast genes that encode proteins involved in the NMD process, UPF1, UPF2, and UPF3, also reduce the efficiency of translation termination. In the current study, we compared the efficiency of translation termination in a upf1Delta strain and a [PSI(+)] strain using a collection of translation termination reporter constructs. The [PSI(+)] state is caused by a prion form of the polypeptide chain release factor eRF3 that limits its availability to participate in translation termination. In contrast, the mechanism by which Upf1p influences translation termination is poorly understood. The efficiency of translation termination is primarily determined by a tetranucleotide termination signal consisting of the stop codon and the first nucleotide immediately 3' of the stop codon. We found that the upf1Delta mutation, like the [PSI(+)] state, decreases the efficiency of translation termination over a broad range of tetranucleotide termination signals in a unique, context-dependent manner. These results suggest that Upf1p may associate with the termination complex prior to polypeptide chain release. We also found that the increase in readthrough observed in a [PSI(+)]/upf1Delta strain was larger than the readthrough observed in strains carrying either defect alone, indicating that the upf1Delta mutation and the [PSI(+)] state influence the termination process in distinct ways. Finally, our analysis revealed that the mRNA destabilization associated with NMD could be separated into two distinct forms that correlated with the extent the premature stop codon was suppressed. The minor component of NMD was a 25% decrease in mRNA levels observed when readthrough was >/=0.5%, while the major component was represented by a larger decrease in mRNA abundance that was observed only when readthrough was 相似文献   

Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). The level of sensitivity of a PTC-containing mRNA to NMD is multifactorial. We have previously shown that human β-globin mRNAs carrying PTCs in close proximity to the translation initiation AUG codon escape NMD. This was called the ‘AUG-proximity effect’. The present analysis of nonsense codons in the human α-globin mRNA illustrates that the determinants of the AUG-proximity effect are in fact quite complex, reflecting the ability of the ribosome to re-initiate translation 3′ to the PTC and the specific sequence and secondary structure of the translated ORF. These data support a model in which the time taken to translate the short ORF, impacted by distance, sequence, and structure, not only modulates translation re-initiation, but also impacts on the exact boundary of AUG-proximity protection from NMD.     

无义介导的mRNA降解(NMD)

无义介导的mRNA降解(nonsense-mediated mRNA decay,NMD）作为真核细胞中重要RNA监控机制,识别并降解开放阅读框中含有提前终止密码子（premature termination codon,PTC）的mRNA,以避免因截短的蛋白产物积累对细胞造成毒害. NMD还调控正常生理基因的表达,暗示其在真核细胞中扮演重要角色. NMD途径的关键是PTC的识别.本文通过3种模型来分别阐述发现于哺乳动物、酵母等不同有机体的识别机制.通常由NMD因子UPF1（up-frameshift）等被招募至含PTC的mRNA上,借助这些因子组装形成“功能复合体”并激活降解.但目前对于PTC识别后的过程仍认识有限,本文通过综述NMD途径的分子机制以更好地理解其生物学意义.