Frameshifting at collided ribosomes is modulated by elongation factor eEF3 and by Integrated Stress Response regulators Gcn1 and Gcn20

Abstract

Ribosome stalls can result in ribosome collisions that elicit quality control responses, one function of which is to prevent frameshifting by the stalled ribosome, an activity that entails interaction of the conserved yeast protein Mbf1 with uS3 on colliding ribosomes. However, the full spectrum of factors that mediate frameshifting during ribosome collisions is unknown. To delineate such factors in the yeast Saccharomyces cerevisiae, we used genetic selections for mutants that either suppress or increase frameshifting from a known ribosome stall site, CGA codon repeats. We show that the general translation elongation factor eEF3 promotes frameshifting, while Integrated Stress Response (ISR) pathway components Gcn1 and Gcn20 suppress frameshifting. We found a mutant form of eEF3 that specifically suppressed frameshifting, but not translation inhibition by CGA codons. Thus, we infer that frameshifting at collided ribosomes requires eEF3, which facilitates tRNA-mRNA translocation and E-site tRNA release in yeast and other single cell organisms. By contrast, we found that removal of either Gcn1 or Gcn20, which bind collided ribosomes with Mbf1, increased frameshifting. Thus, we conclude that frameshifting is suppressed by Gcn1 and Gcn20, although these effects are not mediated through activation of the ISR. Furthermore, we examined the relationship of eEF3-mediated frameshifting to other quality control mechanisms, finding that the eEF3-mediated frameshifting competes with No-Go decay, Mbf1 and Gcn1/20. Thus, these results provide evidence of a direct link between translation elongation and frameshifting at collided ribosomes, as well as evidence that frameshifting competes with other quality control pathways that act on collided ribosomes.

The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress

“…Previous studies showed that an E3 ubiquitin ligase, Hel2, triggers the RQC pathway by ubiquitinating collided ribosomes stalled at positively charged amino acid sequences, such as poly-Arg or poly-Lys (Brandman et al, 2012; Houston et al, 2022; Ikeuchi et al, 2019; Matsuo et al, 2017; Matsuo et al, 2020). Ubiquitination leads to ribosome rescue but, in the absence of Hel2, collided ribosomes bypass these stall-inducing sequences and continue translating.…”

Initiator tRNA lacking 1-methyladenosine is unexpectedly targeted by the rapid tRNA decay pathway in evolutionarily distant yeast species

“…We obtained the original trm6-504 (Y200) and its WT parent (Y190) from Dr. James Anderson. The BY trm6-504 strain was reconstructed essentially as previously described (70), with three DNA components constructed in a plasmid vector: first, nt 893-1434 of the TRM6 coding sequence (containing the C1292G mutation of the trm6-504 mutant) and 204 nt of the 3’ UTR; second, K. lactis URA5 ; third, nt 1384-1434 of the TRM6 coding region. The DNA construct was removed from the vector, transformed into S. cerevisiae WT cells by linear transformation, confirmed by PCR, and then strains were plated onto media containing 5-FOA to select for Ura - cells obtained by homologous recombination, which were sequence verified.…”