A vast evolutionarily transient translatome contributes to phenotype and fitness

Abstract

Ribosome profiling experiments demonstrate widespread translation of eukaryotic genomes outside of annotated protein-coding genes. However, it is unclear how much of this "noncanonical" translation contributes biologically relevant microproteins rather than insignificant translational noise. Here, we developed an integrative computational framework (iRibo) that leverages hundreds of ribosome profiling experiments to detect signatures of translation with high sensitivity and specificity. We deployed iRibo to construct a reference translatome in the model organism S. cerevisiae. We identified ~19,000 noncanonical translated elements outside of the ~5,400 canonical yeast protein-coding genes. Most (65%) of these non-canonical translated elements were located on transcripts annotated as non-coding, or entirely unannotated, while the remainder were located on the 5' and 3' ends of mRNA transcripts. Only 14 non-canonical translated elements were evolutionarily conserved. In stark contrast with canonical protein-coding genes, the great majority of the yeast noncanonical translatome appeared evolutionarily transient and showed no signatures of selection. Yet, we uncovered phenotypes for 53% of a representative subset of evolutionarily transient translated elements. The iRibo framework and reference translatome described here provide a foundation for further investigation of a largely unexplored, but biologically significant, evolutionarily transient translatome.

Pervasive translation in Mycobacterium tuberculosis

“…Some sORFs encode functional small proteins that contribute to cell fitness, whereas other sORFs function as cis -acting regulators. In eukaryotes, there have been reports of ‘pervasive translation’ of thousands of unannotated sORFs, likely due to the imperfect specificity of the translation machinery ( Ingolia et al, 2014 ; Ruiz-Orera et al, 2018 ; Wacholder et al, 2021 ). The function, if any, of these sORFs and their encoded proteins is unclear, although they are rarely subject to purifying selection ( Ruiz-Orera et al, 2018 ; Wacholder et al, 2021 ).…”

“…In eukaryotes, there have been reports of ‘pervasive translation’ of thousands of unannotated sORFs, likely due to the imperfect specificity of the translation machinery ( Ingolia et al, 2014 ; Ruiz-Orera et al, 2018 ; Wacholder et al, 2021 ). The function, if any, of these sORFs and their encoded proteins is unclear, although they are rarely subject to purifying selection ( Ruiz-Orera et al, 2018 ; Wacholder et al, 2021 ). Nonetheless, pervasively translated eukaryotic sORFs may function as ‘proto-genes’, that, over the course of evolution, can acquire a function promoting cell fitness, a process referred to as ‘de novo gene birth’ ( Blevins et al, 2021 ; Carvunis et al, 2012 ; Ruiz-Orera et al, 2018 ; Vakirlis et al, 2018 ; Vakirlis et al, 2020 ).…”

Evolutionary Characterization of the Short Protein SPAAR

“…To accomplish this, all reads were shifted such that a read pattern of triplet periodicity corresponded to the coding sequence of annotated genes, as described in Malone et al 2017 [ 41 ]. The significance of triplet periodicity of Ribo-seq reads was then assessed for the SPAAR ORF in each tissue using the method described in Wacholder et al [ 16 ]. For each codon, the position within the codon that had the most reads was determined.…”

“…The x -axis corresponds to each position in the 237 bp long SPAAR ORF in the platypus, with 1 as the first position. Reads in each platypus tissue show significant triplet periodicity by a binomial test [ 16 ], with a preference for aligning to the first codon position shown in black lines.…”

“…The evolutionary origins of microproteins are of considerable interest. Genome-wide studies find that the unannotated coding sequences identified by ribosome profiling tend to be much less conserved across species than annotated genes [ 7 , 15 , 16 , 17 ]. Among well-characterized microproteins, many lack homologs beyond a narrow taxonomic range.…”