One role of messenger RNA (mRNA) degradation is to maintain the fidelity of gene expression by degrading aberrant transcripts. Recent results show that mRNAs without translation termination codons are unstable in eukaryotic cells. We used yeast mutants to demonstrate that these "nonstop" mRNAs are degraded by the exosome in a 3'-to-5' direction. The degradation of nonstop transcripts requires the exosome-associated protein Ski7p. Ski7p is closely related to the translation elongation factor EF1A and the translation termination factor eRF3. This suggests that the recognition of nonstop mRNAs involves the binding of Ski7p to an empty aminoacyl-(RNA-binding) site (A site) on the ribosome, thereby bringing the exosome to a mRNA with a ribosome stalled near the 3' end. This system efficiently degrades mRNAs that are prematurely polyadenylated within the coding region and prevents their expression.
Translation is an important mechanism to monitor the quality of messenger RNAs (mRNAs), as exemplified by the translation-dependent recognition and degradation of transcripts harboring premature termination codons (PTCs) by the nonsense-mediated mRNA decay (NMD) pathway. We demonstrate in yeast that mRNAs lacking all termination codons are as labile as nonsense transcripts. Decay of "nonstop" transcripts in yeast requires translation but is mechanistically distinguished from NMD and the major mRNA turnover pathway that requires deadenylation, decapping, and 5'-to-3' exonucleolytic decay. These data suggest that nonstop decay is initiated when the ribosome reaches the 3' terminus of the message. We demonstrate multiple physiologic sources of nonstop transcripts and conservation of their accelerated decay in mammalian cells. This process regulates the stability and expression of mRNAs that fail to signal translational termination.
The exosome is a protein complex consisting of a variety of 3-to-5 exonucleases that functions both in 3-to-5 trimming of rRNA precursors and in 3-to-5 degradation of mRNA. To determine additional exosome functions, we examined the processing of a variety of RNAs, including tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), RNase P, RNase MRP, and SRP RNAs, and 5S rRNAs in mutants defective in either the core components of the exosome or in other proteins required for exosome function. These experiments led to three important conclusions. First, exosome mutants accumulate 3-extended forms of the U4 snRNA and a wide variety of snoRNAs, including snoRNAs that are independently transcribed or intron derived. This finding suggests that the exosome functions in the 3 end processing of these species. Second, in exosome mutants, transcripts for U4 snRNA and independently transcribed snoRNAs accumulate as 3-extended polyadenylated species, suggesting that the exosome is required to process these 3-extended transcripts. Third, processing of 5.8S rRNA, snRNA, and snoRNA by the exosome is affected by mutations of the nuclear proteins Rrp6p and Mtr4p, whereas mRNA degradation by the exosome required Ski2p and was not affected by mutations in RRP6 or MTR4. This finding suggests that the cytoplasmic and nuclear forms of the exosome represent two functionally different complexes involved in distinct 3-to-5 processing and degradation reactions.The production of a wide variety of RNA species in eukaryotic cells requires specific 3Ј trimming reactions. Such reactions occur in the processing of a diversity of primary transcripts, including those produced by RNA polymerase I, 5.8S rRNA, 18S rRNA, and 25S rRNA (reviewed in reference 35); those produced by RNA polymerase II, including both small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs); and transcripts produced by RNA polymerase III, including tRNAs (reviewed in reference 10) and 5S rRNA (33). This diversity of substrates raises the issue of how each unique RNA is properly processed to generate a specific mature 3Ј end. In addition, mRNAs are subjected to a variety of 3Ј trimming events, including a specific-length trimming of the poly(A) tail (3), cytoplasmic deadenylation of the mRNA (reviewed in reference 14), and eventual 3Ј-to-5Ј degradation of the body of the transcript in the cytoplasm (23,24). An unresolved question is whether these different 3Ј trimming reactions are carried out by a few relatively general 3Ј-to-5Ј exonucleases, or if there are a large number of specific nucleases that each act on limited subsets of substrates.Recent observations have suggested that at least two distinct 3Ј-to-5Ј exonucleolytic processing events are performed by the same exonuclease complex. This is based on the identification of a multiprotein complex, termed the exosome, that contains at least 10 different polypeptides that have been shown to be 3Ј-to-5Ј exonucleases or have sequence similarity to known 3Ј-to-5Ј exonucleases (1, 22). More important, this co...
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