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 ribosome frameshifting, 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 affect frameshifting from a known ribosome stall site, CGA codon repeats. We show that the general translation elongation factor eEF3 and the Integrated Stress Response (ISR) pathway components Gcn1 and Gcn20 modulate frameshifting in opposing manners. 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 primarily through activation of the ISR. Furthermore, we examined the relationship between eEF3-mediated frameshifting and other quality control mechanisms, finding that Mbf1 requires either Hel2 or Gcn1 to suppress frameshifting with wild type eEF3. Thus, these results provide evidence of a direct link between translation elongation and frameshifting at collided ribosomes, as well as evidence that frameshifting is constrained by quality control mechanisms that act on collided ribosomes.

Initiator tRNA lacking 1-methyladenosine is 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 [ 72 ], 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.…”

“…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 [72], 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 Uracells obtained by homologous recombination, which were sequence verified.…”

Two modes of Cue2-mediated mRNA cleavage with distinct substrate recognition initiate no-go decay

“…Mbf1 is associated with the colliding ribosome and acts with ribosomal proteins Rps3/uS3 and Asc1/RACK1 to prevent frameshifting at inhibitory CGA–CGA codon pairs in yeast ( 23 , 24 ). However, there is no direct evidence to show the essential roles of Mbf1 in RQC and NGD.…”

“…An additional player in the context of ribosome collisions is the multiprotein bridging factor 1 (Mbf1) which is associated with the colliding ribosome and acts with ribosomal proteins Rps3/uS3 and Asc1/RACK1 to prevent frameshifting at inhibitory CGA–CGA codon pairs in yeast ( 23 , 24 ). It has been proposed that Mbf1 maintains the reading frame during the translation of rare codons.…”

Inducible Systemic Gcn1 Deletion in Mice Leads to Transient Body Weight Loss upon Tamoxifen Treatment Associated with Decrease of Fat and Liver Glycogen Storage

“…In fact, cryo-electron microscopic demonstration of yeast GCN1 that is directly bound to a disome (two ribosomes colliding) indicated the role of GCN1 during translation stress in recognizing ribosome collision [ 11 ]. In addition, yeast GCN1 suppresses frameshift at the ribosome stall site of the CGA codon repeat [ 12 ]. In terms of the regulation of translational elongation, GCN2 plays important roles in protecting the mice with rare codon-mediated neuronal degeneration [ 13 ] and in rare codon-mediated translational feedback in neurospora [ 14 ], although the role of GCN1 in these situations was not established.…”