Quantification of elongation stalls and impact on gene expression in yeast

Abstract

Ribosomal pauses are a critical part of co-translational events including protein folding and localization. However, extended ribosome pauses can lead to ribosome collisions, resulting in the activation of ribosome rescue pathways and turnover of protein and mRNA. While this relationship has been known, there has been little exploration of how ribosomal stalls impact translation duration at a quantitative level. We have taken a method used to measure elongation time and adapted it for use in S. cerevisiae to quantify the impact of elongation stalls. We find, in transcripts containing Arg CGA codon repeat-induced stalls, a Hel2-mediated dose-dependent decrease in protein expression and mRNA level and an elongation delay on the order of minutes. In transcripts that contain synonymous substitutions to non-optimal Leu codons, there is a decrease in protein and mRNA levels, as well as similar elongation delay, but this occurs through a non-Hel2-mediated mechanism. Finally, we find that Dhh1 selectively increases protein expression, mRNA level, and elongation rate. This indicates that distinct poorly translated codons in an mRNA will activate different rescue pathways despite similar elongation stall durations. Taken together, these results provide new quantitative mechanistic insight into the surveillance of translation and the roles of Hel2 and Dhh1 in mediating ribosome pausing events.

Is there a localized role for translational quality control?

“…This pathway is triggered when the E3 ubiquitin ligase, ZNF598 (Hel2 in yeast), ubiquitinates specific ribosomal proteins within a disome complex ( Brandman et al 2012 ; Juszkiewicz and Hegde 2017 ; Matsuo et al 2017 ; Sundaramoorthy et al 2017 ). The ubiquitination event prevents the stalled ribosome from resuming translation and leads to its rescue (removal from the mRNA) by the RQC trigger complex that includes ASCC3 (Slh1 in yeast) ( Matsuo et al 2017 ; Juszkiewicz et al 2020b ; Matsuo et al 2020 ; Hou et al 2023 ). The peptidyl-tRNA remains associated with the 60S subunit and is then targeted for degradation, a process involving many proteins, including LISTERIN and NEMF, and includes the addition of carboxy-terminal Ala and Thr residues that serve as a degron signal (CAT-tails).…”