Disome-seq reveals widespread ribosome collisions that recruit co-translational chaperones

Abstract

Regulation of translation elongation plays a crucial role in determining absolute protein levels and ensuring the correct localization and folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes (ribo-seq). However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation. In this study, we performed disome-seq to sequence mRNA fragments protected by two stacked ribosomes -a product of translational pauses during which the 5′-ribosome collides with the 3′-paused one. We detected widespread ribosome collisions that are missed in traditional ribo-seq. These collisions are due to 1) slow ribosome release when stop codons are at the A-site, 2) slow peptide bond formation from proline, glycine, asparagine, and cysteine when they are at the P-site, and 3) slow leaving of polylysine from the exit tunnel of ribosomes.The paused ribosomes can continue translating after collisions, as suggested by the structure of disomes obtained by cryo-electron microscopy (cryo-EM). Collided ribosomes recruit chaperones, which can aid in the co-translational folding of the nascent peptides. Therefore, cells use regulated ribosome collisions to ensure protein homeostasis.

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

“…showing that short polybasic sequences promote ribosome collisions and are enriched in the ribosome exit tunnel of the disomes [29]. The authors argue that the translation of these disomes is likely to resume without recruiting the RQC pathway and, they hypothesize that these polybasic-induced pauses (and collisions) can favor the co-translational folding of upstream sequences [29]. Recently, the yeast gene SDD1 was identified as a natural target of RQC.…”

“…Recently, it was shown that ribosomes with open A sites produce shorter mRNA fragments (20-22 nucleotides) than ribosomes with occupied A sites, which produce the longer mRNA fragments (27)(28)(29) nucleotides) analyzed in Figure 3a [35]. Structural and biochemical evidence demonstrated that the electrostatic repulsion caused by the presence of a polybasic sequence inside the exit tunnel can decrease the binding efficiency of an incoming tRNA carrying an additional positively charged residue, leading to empty A site ribosome [25,39,68].…”

“…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].…”

“…It has been proposed that the trigger for RQC activity is ribosome collision, with the formation of disomes, trisomes, and even tetrasomes [14,[25][26][27][28]. However, recent studies on the global analysis of disome formation suggested that collisions are relatively common, and most of these events are resolved without translation termination [29]. What determines whether a collision event will become a target of RQC or not is still an open question, but the disome structure, the time required to resume translation, and/or exposure of an empty A site, seem to play an important role in this decision [30].…”