The specification and patterning of vulval precursor cells (VPCs) in Caenorhabditiselegans is achieved using a conserved EGFR/RAS signaling pathway that is activated by the ligand lin-3/EGF, which is secreted by the neighboring somatic gonad. Previous work has demonstrated that the expression of lin-3 must be tightly regulated to ensure that only three of six equivalent VPCs are induced to differentiate into the mature vulva. Here, we have identified a novel regulator of EGFR/RAS signaling, let-765/nsh-1, that functions upstream of the pathway to promote vulval induction. let-765 encodes a conserved DExD/H box helicase protein and is the C. elegans ortholog of Drosophila strawberry notch. By investigating genetic interactions between let-765 and RAS pathway genes as well as with synthetic multivulva (synMuv) genes, we have demonstrated that let-765 positively regulates the RAS pathway and antagonizes synMuv activity at the level of lin-3/EGF. In support of these proposals, we found that LET-765 is required for producing wild-type levels of lin-3 mRNA. Mutations in let-765 result in pleiotropic phenotypes that imply its function must be required in multiple developmental processes and, together with data presented here, suggest that LET-765 promotes the expression of diverse targets, potentially through interactions with transcriptional activator or repressor complexes.

During translation, an mRNA is typically occupied by multiple ribosomes sparsely distributed across the coding sequence. This distribution, mediated by slow rates of initiation relative to elongation, ensures that they rarely collide with each other, but given the stochastic nature of protein synthesis, collision events do occur. Recent work from our lab suggested that collisions signal for mRNA degradation through no-go decay (NGD). We have explored the impact of stalling on ribosome function when NGD is compromised and found it to result in +1 frameshifting. We used reporters that limit the number of ribosomes on a transcript to show that +1 frameshifting is induced through ribosome collision in yeast and bacteria. Furthermore, we observe a positive correlation between ribosome density and frameshifting efficiency. It is thus tempting to speculate that NGD, in addition to its role in mRNA quality control, evolved to cope with stochastic collision events to prevent deleterious frameshifting events.

No-go Decay (NGD) is a process that has evolved to deal with stalled ribosomes resulting from structural blocks or aberrant mRNAs. The process is distinguished by an endonucleolytic cleavage prior to degradation of the transcript. While many of the details of the pathway have been described, the identity of the endonuclease remains unknown. Here we identify residues of the small subunit ribosomal protein Rps3 that are important for NGD by affecting the cleavage reaction. Mutation of residues within the ribosomal entry tunnel that contact the incoming mRNA leads to significantly reduced accumulation of cleavage products, independent of the type of stall sequence, and renders cells sensitive to damaging agents thought to trigger NGD. These phenotypes are distinct from those seen in combination with other NGD factors, suggesting a separate role for Rps3 in NGD. Conversely, ribosomal proteins ubiquitination is not affected by rps3 mutations, indicating that upstream ribosome quality control (RQC) events are not dependent on these residues. Together, these results suggest that Rps3 is important for quality control on the ribosome and strongly supports the notion that the ribosome itself plays a central role in the endonucleolytic cleavage reaction during NGD.

No-go decay (NGD) is a eukaryotic quality control mechanism that evolved to cope with translational arrests. The process is characterized by an endonucleolytic cleavage near the stall sequence, but the mechanistic details are unclear. Our analysis of cleavage sites indicates that cleavage requires multiple ribosomes on the mRNA. We also show that reporters harboring stall sequences near the initiation codon, which cannot accommodate multiple ribosomes, are not subject to NGD. Consistent with our model, we uncover an inverse correlation between ribosome density per mRNA and cleavage efficiency. Furthermore, promoting global ribosome collision in vivo resulted in ubiquitination of ribosomal proteins, suggesting that collision is sensed by the cell to initiate downstream quality control processes. Collectively, our data suggest that NGD and subsequent quality control are triggered by ribosome collision. This model provides insight into the regulation of quality control processes and the manner by which they reduce off-target effects.

Termination of protein synthesis on the ribosome is catalyzed by release factors (RFs), which share a conserved glycine-glycine-glutamine (GGQ) motif. The glutamine residue is methylated in vivo, but a mechanistic understanding of its contribution to hydrolysis is lacking. Here, we show that the modification, apart from increasing the overall rate of termination on all dipeptides, substantially increases the rate of peptide release on a subset of amino acids. In the presence of unmethylated RFs, we measure rates of hydrolysis that are exceptionally slow on proline and glycine residues and approximately two orders of magnitude faster in the presence of the methylated factors. Structures of 70S ribosomes bound to methylated RF1 and RF2 reveal that the glutamine side-chain methylation packs against 23S rRNA nucleotide 2451, stabilizing the GGQ motif and placing the side-chain amide of the glutamine toward tRNA. These data provide a framework for understanding how release factor modifications impact termination.

Oxidation and alkylation of nucleobases are known to disrupt their base-pairing properties within RNA. It is, however, unclear whether organisms have evolved general mechanism(s) to deal with this damage. Here we show that the mRNA-surveillance pathway of no-go decay and the associated ribosome-quality control are activated in response to nucleobase alkylation and oxidation. Our findings reveal that these processes are important for clearing chemically modified mRNA and the resulting aberrant-protein products. In the absence of Xrn1, the level of damaged mRNA significantly increases. Furthermore, deletion of LTN1 results in the accumulation of protein aggregates in the presence of oxidizing and alkylating agents. This accumulation is accompanied by Hel2-dependent regulatory ubiquitylation of ribosomal proteins. Collectively, our data highlight the burden of chemically damaged mRNA on cellular homeostasis and suggest that organisms evolved mechanisms to counter their accumulation.