Aberrant mRNAs with extended 3' UTRs are substrates for rapid degradation by mRNA surveillance

The mRNA surveillance system is known to rapidly degrade aberrant mRNAs that contain premature termination codons in a process referred to as nonsense-mediated decay. A second class of aberrant mRNAs are those wherein the 3' UTR is abnormally extended due to a mutation in the polyadenylation site. We provide several observations that these abnormally 3'-extended mRNAs are degraded by the same machinery that degrades mRNAs with premature nonsense codons. First, the decay of the 3'-extended mRNAs is dependent on the same decapping enzyme and 5'-to-3' exonuclease. Second, the decay is also dependent on the proteins encoded by the UPF1, UPF2, and UPF3 genes, which are known to be specifically required for the rapid decay of mRNAs containing nonsense codons. Third, the ability of an extended 3' UTR to trigger decay is prevented by stabilizing sequences within the PGK1 coding region that are known to protect mRNAs from the rapid decay induced by premature nonsense codons. These results indicate that the mRNA surveillance system plays a role in degrading abnormally extended 3' UTRs. Based on these results, we propose a model in which the mRNA surveillance machinery degrades aberrant mRNAs due to the absence of the proper spatial arrangement of the translation-termination codon with respect to the 3' UTR element as defined by the utilization of a polyadenylation site.     

Translation of aberrant mRNAs lacking a termination codon or with a shortened 3'-UTR is repressed after initiation in yeast

A novel mRNA surveillance for mRNA lacking a termination codon (nonstop mRNA) has been proposed in which Ski7p is thought to recognize stalled ribosomes at the 3' end of mRNA. Here we report our analysis of translation and decay of nonstop mRNAs in Saccharomyces cerevisiae. Although the reduction of nonstop mRNAs was only 4.5-fold, a level that is sufficient for residual protein synthesis, translation products of nonstop mRNAs were hardly detectable. We show that nonstop mRNAs were associated with polysomes, but not with Pab1p. We also show that ribosomes translating nonstop mRNA formed stable and heavy polysome complexes with mRNA. These data suggest that ribosome stalling at the 3' end of nonstop mRNA may block further rounds of translation, hence repressing protein synthesis. Furthermore, it was found that the 5' --> 3' decay pathway was accelerated for nonstop mRNA decay in the absence of Ski7p. We also found that translation of aberrant mRNAs with a shortened 3'-UTR was repressed, suggesting that an improper spatial distance between the termination codon and the 3' end of mRNA results in translation repression.     

tmRNA determinants required for facilitating nonstop mRNA decay

In bacteria, ribosomes stalled on nonstop mRNAs are rescued by tmRNA in a unique process called trans-translation. The two known tmRNA functions in trans-translation are (1) a tRNA-like function, which transfers the partially synthesized protein fragment to itself; and (2) an mRNA-like function, which enables ribosomes to resume and terminate translation on tmRNA as a surrogate template. We present evidence to demonstrate that tmRNA performs a third function, namely, facilitating the degradation of the causative defective mRNA. Our investigations have revealed the identity of key sequence determinants that promote the degradation of the nonstop mRNA. These sequence determinants are located in the distal part of the tmRNA open reading frame, encoding the ultimate, penultimate, and anti-penultimate amino acids of the peptide tag. We show that mutation of these tmRNA sequence elements has an adverse affect on the disposal of the nonstop mRNA, while leaving the tRNA and mRNA functions entirely unaffected. More significantly, specific mutations that change the nucleotide sequence of the peptide-reading frame without altering the nature or identity of the encoded amino acids still exhibit the characteristic defect in nonstop mRNA decay. In contrast, mutations in codons 3, 4, 5, and 6 of the tmRNA open reading frame do not have an adverse affect on degradation of defective mRNAs. Based on these results, we propose that tmRNA plays an important role in promoting the decay of nonstop mRNAs and that sequence elements in the distal segment of the peptide-reading frame make sequence-specific contributions that are crucial for this activity.     

Dom34:hbs1 plays a general role in quality-control systems by dissociation of a stalled ribosome at the 3' end of aberrant mRNA

Translation arrest leads to an endonucleolytic cleavage of mRNA that is termed no-go decay (NGD). It has been reported that the Dom34:Hbs1 complex stimulates this endonucleolytic cleavage of mRNA induced by translation arrest in vivo and dissociates subunits of a stalled ribosome in vitro. Here we report that Dom34:Hbs1 dissociates the subunits of a ribosome that is stalled at the 3' end of mRNA in vivo, and has a crucial role in both NGD and nonstop decay. Dom34:Hbs1-mediated dissociation of a ribosome that is stalled at the 3' end of mRNA is required for degradation of a 5'-NGD intermediate. Dom34:Hbs1 facilitates the decay of nonstop mRNAs from the 3' end by exosomes and is required for the complete degradation of nonstop mRNA decay intermediates. We propose that Dom34:Hbs1 stimulates degradation of the 5'-NGD intermediate and of nonstop mRNA by dissociating the ribosome that is stalled at the 3' end of the mRNA.     

Quality control systems for aberrant mRNAs induced by aberrant translation elongation and termination

RNA processing is an essential gene expression step and plays a crucial role to achieve diversity of gene products in eukaryotes. Various aberrant mRNAs transiently produced during RNA processing reactions are recognized and eliminated by specific quality control systems. It has been demonstrated that these mRNA quality control systems stimulate the degradation of aberrant mRNA to prevent the potentially harmful products derived from aberrant mRNAs. Recent studies on quality control systems induced by abnormal translation elongation and termination have revealed that both aberrant mRNAs and proteins are subjected to rapid degradation. In NonStop Decay (NSD) quality control system, a poly(A) tail of nonstop mRNA is translated and the synthesis of poly-lysine sequence results in translation arrest followed by co-translational degradation of aberrant nonstop protein. In No-Go Decay (NGD) quality control system, the specific amino acid sequences of the nascent polypeptide induce ribosome stalling, and the arrest products are ubiquitinated and rapidly degraded by the proteasome. In Nonfunctional rRNA Decay (NRD) quality control system, aberrant ribosomes composed of nonfunctional ribosomal RNAs are also eliminated when aberrant translation elongation complexes are formed on mRNA. I describe recent progresses on the mechanisms of quality control systems and the relationships between quality control systems. This article is part of a Special issue entitled: RNA Decay mechanisms.     

Lon protease degrades transfer-messenger RNA-tagged proteins

Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with lambda-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.