Nonsense-mediated messenger RNA decay (NMD) is triggered by premature translation termination, but the features distinguishing premature from normal termination are unknown. One model for NMD suggests that decay-inducing factors bound to mRNAs during early processing events are routinely removed by elongating ribosomes but remain associated with mRNAs when termination is premature, triggering rapid turnover. Recent experiments challenge this notion and suggest a model that posits that mRNA decay is activated by the intrinsically aberrant nature of premature termination. Here we use a primer extension inhibition (toeprinting) assay to delineate ribosome positioning and find that premature translation termination in yeast extracts is indeed aberrant. Ribosomes encountering premature UAA or UGA codons in the CAN1 mRNA fail to release and, instead, migrate to upstream AUGs. This anomaly depends on prior nonsense codon recognition and is eliminated in extracts derived from cells lacking the principal NMD factor, Upf1p, or by flanking the nonsense codon with a normal 3'-untranslated region (UTR). Tethered poly(A)-binding protein (Pab1p), used as a mimic of a normal 3'-UTR, recruits the termination factor Sup35p (eRF3) and stabilizes nonsense-containing mRNAs. These findings indicate that efficient termination and mRNA stability are dependent on a properly configured 3'-UTR.

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In addition to their well-documented roles in the promotion of nonsense-mediated mRNA decay (NMD), yeast Upf proteins (Upf1, Upf2/Nmd2, and Upf3) also manifest translational regulatory functions, at least in vitro, including roles in premature translation termination and subsequent reinitiation. Here, we find that all upfD strains also fail to reinitiate translation after encountering a premature termination codon (PTC) in vivo, a result that led us to seek a unifying mechanism for all of these translation phenomena. Comparisons of the in vitro translational activities of wild-type (WT) and upf1D extracts were utilized to test for a Upf1 role in post-termination ribosome reutilization. Relative to WT extracts, non-nucleased extracts lacking Upf1 had approximately twofold decreased activity for the translation of synthetic CAN1/LUC mRNA, a defect paralleled by fewer ribosomes per mRNA and reduced efficiency of the 60S joining step at initiation. These deficiencies could be complemented by purified FLAG-Upf1, or 60S subunits, and appeared to reflect diminished cycling of ribosomes from endogenous PTC-containing mRNAs to exogenously added synthetic mRNA in the same extracts. This hypothesis was tested, and supported, by experiments in which nucleased WT or upf1D extracts were first challenged with high concentrations of synthetic mRNAs that were templates for either normal or premature translation termination and then assayed for their capacity to translate a normal mRNA. Our results indicate that Upf1 plays a key role in a mechanism coupling termination and ribosome release at a PTC to subsequent ribosome reutilization for another round of translation initiation.

NMD (nonsense-mediated mRNA decay) is a cellular quality-control mechanism in which an otherwise stable mRNA is destabilized by the presence of a premature termination codon. We have defined the set of endogenous NMD substrates, demonstrated that they are available for NMD at every round of translation, and showed that premature termination and normal termination are not equivalent biochemical events. Premature termination is aberrant, and its NMD-stimulating defects can be reversed by the presence of tethered poly(A)-binding protein (Pab1p) or tethered eRF3 (eukaryotic release factor 3) (Sup35p). Thus NMD appears to be triggered by a ribosome's failure to terminate adjacent to a properly configured 3'-UTR (untranslated region), an event that may promote binding of the UPF/NMD factors to stimulate mRNA decapping.

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Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

RNA helicases are involved in almost every aspect of RNA metabolism, yet very little is known about the regulation of this class of enzymes. In Saccharomyces cerevisiae, the stability and translational fidelity of nonsense-containing mRNAs are controlled by the group I RNA helicase Upf1 and the proteins it interacts with, Upf2 and Upf3. Combining the yeast two-hybrid system with genetic analysis, we show here that the cysteine-and histidine-rich (CH) domain and the RNA helicase domain of yeast Upf1 can engage in two new types of molecular interactions: an intramolecular interaction between these two domains and self-association of each of these domains. Multiple observations indicate that these molecular interactions are crucial for Upf1 regulation. First, coexpression of the CH domain and the RNA helicase domain in trans can reconstitute Upf1 function in both promoting nonsense-mediated mRNA decay (NMD) and preventing nonsense suppression. Second, mutations that disrupt Upf1 intramolecular interaction cause loss of Upf1 function. These mutations weaken Upf2 interaction and, surprisingly, promote Upf1 selfassociation. Third, the genetic defects resulting from deficiency in Upf1 intramolecular interaction or RNA binding are suppressed by expression of Upf2. Collectively, these data reveal a set of sequential molecular interactions and their roles in regulating Upf1 function during activation of NMD and suggest that cis intramolecular interaction and trans self-association may be general mechanisms for regulation of RNA helicase functions. E ukaryotic cells have evolved multiple quality control mechanisms to ensure the fidelity of gene expression (1-3). One of these mechanisms, nonsense-mediated mRNA decay (NMD), which operates during mRNA translation, targets transcripts containing a premature termination codon (PTC) (4). This mRNA decay pathway ensures rapid degradation of PTC-containing transcripts and thus prevents the cell from accumulating truncated and potentially deleterious polypeptides (5, 6). NMD also targets a subset of functionally relevant wild-type mRNAs (7-9), suggesting that this decay pathway has a substantial role in posttranscriptional gene regulation and likely controls important cellular functions.From yeast to humans, NMD requires a set of conserved regulatory factors, the Upf proteins: Upf1, Upf2, and Upf3 (4, 7). These factors interact with each other and appear to constitute the core NMD machinery in eukaryotic cells (10-13). Deletion or silencing of each of the genes encoding these factors selectively stabilizes PTC-containing transcripts and other NMD substrates (9, 11, 13-15). In multicellular organisms, NMD also requires additional regulatory factors, including Smg1 and Smg5 to Smg7 (4, 7). These factors control Upf1 phosphorylation and dephosphorylation, a cycle that, in turn, controls several important Upf1 functions during NMD, including translation repression (16), remodeling of terminating messenger ribonucleoprotein particles (mRNPs) (17), and recruitment of the decay enzymes (1...

Ribosomes associated with nonsense-mediated decay factors Upf1, Upf2, or Upf3 were purified by immunoprecipitation, and enrichment and stoichiometry of Upf factors and ribosomal proteins were analyzed by western blot and mass spectrometry. Using a small RNA library preparation protocol that eliminates in-gel RNA and cDNA size selection and incorporates four random nucleotides on each side of the ribosome-protected RNA fragment allowed recovery, detection, and analysis of all size classes of protected fragments from a sample simultaneously.