Transcriptome-wide sites of collided ribosomes reveal sequence determinants of translational pausing in vivo

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. We find pause sites associated with specific codons, amino acids, and 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 and unexpected insights into gene regulation occurring at the step of translation elongation. Six biological samples (3 timepoints x 2 replicates) from GSE67305 were used. Briefly, mice were entrained under 12hours light:dark conditions for 2 weeks and also collected under 12hours light:dark. Mice were sacrificed at 0, 2, and 12h during the 24 hours daily cycle. Two replicates per time point, each replicate is a pool of 2 livers (2 mice).