Release Factor eRF3 Mediates Premature Translation Termination on Polylysine-Stalled Ribosomes in Saccharomyces cerevisiae

Abstract

bRibosome stalling is an important incident enabling the cellular quality control machinery to detect aberrant mRNA. Saccharomyces cerevisiae Hbs1-Dom34 and Ski7 are homologs of the canonical release factor eRF3-eRF1, which recognize stalled ribosomes, promote ribosome release, and induce the decay of aberrant mRNA. Polyadenylated nonstop mRNA encodes aberrant proteins containing C-terminal polylysine segments which cause ribosome stalling due to electrostatic interaction with the ribosomal exit tunnel. Here we describe a novel mechanism, termed premature translation termination, which releases C-terminally truncated translation products from ribosomes stalled on polylysine segments. Premature termination during polylysine synthesis was abolished when ribosome stalling was prevented due to the absence of the ribosomal protein Asc1. In contrast, premature termination was enhanced, when the general rate of translation elongation was lowered. The unconventional termination event was independent of Hbs1-Dom34 and Ski7, but it was dependent on eRF3. Moreover, premature termination during polylysine synthesis was strongly increased in the absence of the ribosome-bound chaperones ribosome-associated complex (RAC) and Ssb (Ssb1 and Ssb2). On the basis of the data, we suggest a model in which eRF3-eRF1 can catalyze the release of nascent polypeptides even though the ribosomal A-site contains a sense codon when the rate of translation is abnormally low.

Increased ribosome density associated to positively charged residues is evident in ribosome profiling experiments performed in the absence of translation inhibitors

“…Polybasic proteins cause similar effects, as demonstrated by our analysis and by different groups. [4][5][6][25][26][27] Our analysis, together with other recent studies exploring the effect of CHX on ribosome profiling, 23,28 showed, that the RP methodology can be more faithfully used to explore ribosomal translation if performed in the absence of translational inhibitors such as cycloheximide and anisomycin. We demonstrated that drug-free RP datasets tend to agree with the experimental evidence obtained by other methods analyzing the role of positively charged amino acids in reducing protein translation.…”

Distinct types of translation termination generate substrates for ribosome-associated quality control

“…Intriguingly, there is prior evidence that Sup45–Sup35 may be capable of binding to stalled ribosomal complexes that contain a sense codon in the A site (63,64,28). Another interesting question concerns the activity of Ltn1 on 60S·NC substrates in the absence of tRNA or Rqc2.…”

‘2A‐Like’ Signal Sequences Mediating Translational Recoding: A Novel Form of Dual Protein Targeting

“…Although no stop codon is involved, eukaryotic translation release (termination) factors 1 and 3 (eRF1/eRF3) are proposed to release the nascent protein from the ribosome, thereby forming the C-terminus of 2A ( Figure 2). In support of this model, eRF3 was recently shown to mediate termination of translation of yeast ribosomes stalled on polylysine tracts (17), although other recent analyses using re-constituted translation systems did not support the involvement of eRFs 1 and 3 (18). Our model of this translational recoding event predicts that three alternative outcomes arise; having synthesized sequences upstream of 2A, ribosomes either (i) resume translation of the downstream sequences, (ii) translation is and 'cleaved' [GFP-2A] plus GUS translation products.…”