Disome and Trisome Profiling Reveal Genome-wide Targets of Ribosome Quality Control

Abstract

The ribosome-associated protein quality control (RQC) system that resolves stalled translation events is activated when ribosomes collide and form disome, trisome, or higher-order complexes. However, it is unclear whether this system distinguishes collision complexes formed on defective mRNAs from those with functional roles on endogenous transcripts. Here, we performed disome and trisome footprint profiling in yeast and found collisions were enriched on diverse sequence motifs known to slow translation. When 60S recycling was inhibited, disomes accumulated at stop codons and could move into the 3 0 UTR to reinitiate translation. The ubiquitin ligase and RQC factor Hel2/ZNF598 generally recognized collisions but did not induce degradation of endogenous transcripts. However, loss of Hel2 triggered the integrated stress response, via phosphorylation of eIF2a, thus linking these pathways. Our results suggest that Hel2 has a role in sensing ribosome collisions on endogenous mRNAs, and such events may be important for cellular homeostasis.

High-throughput 5’P sequencing enables the study of degradation-associated ribosome stalls

“…Termination pause scores were calculated in each dataset by dividing the peaks from termination versus the upstream 27-nt regions (before the following ribosome queuing). Collision scores were calculated from Meydan et al (25) by dividing termination pause scores in disome versus monosome profiling. Dataset for S. cerevisiae tRNA adaptation index, mRNA codon stability index, translation efficiency were obtained from Carneiro et al (26), gene expression level and mRNA half-life were obtained from Xu et al (38), Celik et al (39) and Presnyak et al (10), respectively.…”

Rqc1 and other yeast proteins containing highly positively charged sequences are not targets of the RQC complex

“…For Figure 4, the ribosome profiling of each gene was downloaded from Trips-Viz by aggregating all experiments in the database [50]. Disome Profiling Data: The datasets we used for disome profiling were from D'Orazio et al, 2019 [36] (GEO accession GSE129128), and Meydan and Guydosh, 2020 [37] (GEO accession GSE139036).…”

“…Additionally, we looked directly at the presence of ribosome collisions through the analyses of disome profiling. The disome profiling allows sequencing of mRNA fragments protected by two stacked ribosomes and shows stalling patterns undetected by traditional RP [29,36,37]. Disome footprints could be detected as a sharp peak immediately after the ICPs sequences (Supplementary Figure S3a-c).…”

“…This observation supports the hypothesis that the arrest is caused by electrostatic interactions of the nascent positively charged polypeptide with the ribosome exit tunnel. Since disomes were detected in most transcripts, it is unlikely that the formation of these disomes can trigger the RQC activation [29,37].…”

“…In this work, we first performed a bioinformatic screening to identify the yeast proteins containing polybasic sequences (sequences with the highest concentrations of K/R of the yeast proteome) Then, we analyzed previously published ribosome profiling experiments [25,[34][35][36][37] to access the impact of polybasic sequences on the translation of these proteins. Ribosome profiling is based on the deep sequencing of ribosome-protected mRNA fragments [38].…”