Paper 1: Genome-wide survey of ribosome collision. Paper 2: Disome-seq reveals sequence-mediated coupling of translational pauses and protein structures. Paper 3: Transcriptome-wide sites of collided ribosomes reveal principles of translational pausing

Abstract 1

In protein synthesis, ribosome movement is not always smooth and is rather often impeded for numerous reasons. Although the deceleration of the ribosome defines the fates of the mRNAs and synthesizing proteins, fundamental issues remain to be addressed, including where ribosomes pause in mRNAs, what kind of RNA/amino acid sequence causes this pause, and the physiological significance of this slowdown of protein synthesis. Here, we surveyed the positions of ribosome collisions caused by ribosome pausing in humans and zebrafish on a genome-wide level using modified ribosome profiling. The collided ribosomes, i.e., disomes, emerged at various sites: the proline-proline-lysine motif, stop codons, and the 3′ untranslated region (UTR). The number of ribosomes involved in a collision is not limited to two, but rather four to five ribosomes can form a queue of ribosomes. In particular, XBP1, a key modulator of the unfolded protein response, shows striking queues of collided ribosomes and thus acts as a substrate for ribosome-associated quality control (RQC) to avoid the accumulation of undesired proteins in the absence of stress. Our results provide an insight into the causes and consequences of ribosome slowdown by dissecting the specific architecture of ribosomes.

ABSTRACT 2

The regulation of translation elongation plays a vital role in protein folding; an adequate translational pause provides time and cellular environments for the co-translational folding of nascent peptides. However, the genomic landscape, sequence determinants, and molecular consequences of translational pausing remain mostly unknown. In this study, we performed disome-seq that sequenced mRNA fragments protected by two consecutive ribosomes – a product of severe translational pauses during which the upstream ribosome collides into the paused one. We detected severe translational pauses on ∼75% of yeast genes. These pauses were often explained by one of the three mechanisms: 1) slow ribosome releasing at stop codons, 2) slow peptide formation from proline, glycine, asparagine, and cysteine, and 3) slow leaving of polylysine from the exit tunnel of ribosomes. Notably, these amino acids also terminate the α-helical conformation. Such dual roles of amino acids establish an inborn coupling between the synthetic completion of a structural motif and a translational pause. Furthermore, paused ribosomes often recruit chaperones to assist protein folding. As a consequence, emergent protein structures during evolution should be ready to be correctly folded. Collectively, our study shows widespread translational pauses and sheds lights on a better understanding of the regulation of co-translational protein folding.

Abstract 3

Translation initiation is considered overall rate-limiting for protein biosynthesis, whereas the impact of non-uniform ribosomal elongation rates is largely unknown. Using a modified ribosome profiling protocol based on footprints from two closely packed ribosomes (disomes), we have mapped ribosomal collisions transcriptome-wide in mouse liver. We uncover that the stacking of an elongating onto a paused ribosome occurs frequently and scales with translation rate, trapping ∼10% of translating ribosomes in the disome state. A distinct class of pause sites, independent of translation rate, is indicative of deterministic pausing signals. Pause sites are associated with specific amino acids, peptide motifs, and with structural features of the nascent polypeptide, suggestive of programmed pausing as a widespread mechanism associated with protein folding. Evolutionary conservation at disome sites and experiments indicate functional relevance of translational pausing. Collectively, our disome profiling approach allows novel, unexpected insights into gene regulation occurring at the step of translation elongation.