Identification of novel translated small ORFs in Escherichia coli using complementary ribosome profiling approaches

Abstract

Small proteins of <51 amino acids are abundant across all domains of life but are often overlooked because their small size makes them difficult to predict computationally, and they are refractory to standard proteomic approaches. Ribosome profiling has been used to infer the existence of small proteins by detecting the translation of the corresponding open reading frames (ORFs). Detection of translated short ORFs by ribosome profiling can be improved by treating cells with drugs that stall ribosomes at specific codons. Here, we combine the analysis of ribosome profiling data for Escherichia coli cells treated with antibiotics that stall ribosomes at either start or stop codons. Thus, we identify ribosome-occupied start and stop codons for ~400 novel putative ORFs with high sensitivity. The newly discovered ORFs are mostly short, with 365 encoding proteins of <51 amino acids. We validate translation of several selected short ORFs, and show that many likely encode unstable proteins. Moreover, we present evidence that most of the newly identified short ORFs are not under purifying selection, suggesting they do not impact cell fitness, although a small subset have the hallmarks of functional ORFs.

Pervasive translation in Mycobacterium tuberculosis

“…Indeed, there are many intragenic promoters in Mtb ( Cortes et al, 2013 ; Shell et al, 2015 ), providing an additional source of potential spurious translation. We speculate that like spurious transcripts, which are rapidly degraded by RNases, the protein products of pervasive translation are rapidly degraded, as has been proposed for pervasively translated ORFs in E. coli ( Stringer et al, 2021 ). Since Ribo-seq and Ribo-RET detect translation, not the protein product, the stability of the encoded proteins would not impact our ability to detect the corresponding ORFs.…”

“…Hence, they often fail to identify non-canonical ORFs, including overlapping ORFs ( Burge and Karlin, 1998 ), leaderless ORFs ( Beck and Moll, 2018 ; Lomsadze et al, 2018 ), and short ORFs (sORFs; encoding small proteins of 50 or fewer amino acids; most algorithms have a lower size limit of 50 codons). Recent studies have revealed hundreds of sORFs in diverse bacterial species ( Orr et al, 2020 ; Sberro et al, 2019 ; Storz et al, 2014 ; Stringer et al, 2021 ; VanOrsdel et al, 2018 ; Weaver et al, 2019 ). Some sORFs encode functional small proteins that contribute to cell fitness, whereas other sORFs function as cis -acting regulators.…”

“…Thus, Ribo-RET facilitates the identification of overlapping ORFs by limiting the signal to the start codons ( Meydan et al, 2018 ; Meydan et al, 2019 ). Ribo-RET was recently applied to E. coli , revealing start codons for many previously undescribed ORFs ( Meydan et al, 2019 ; Stringer et al, 2021 ; Weaver et al, 2019 ), including sORFs, and ORFs positioned in frame with annotated ORFs, such that the translated protein is an isoform of the previously described protein. Here, we use a combination of Ribo-seq and Ribo-RET to map translated ORFs in Mtb .…”

Spotlight on alternative frame coding: Two long overlapping genes in Pseudomonas aeruginosa are translated and under purifying selection

“…Antisense transcription, reported in diverse bacteria including P. aeruginosa , often plays a role in gene regulation, indicated by a negative correlation between antisense and mRNA levels ( Dornenburg et al., 2010 ; Eckweiler and Häussler, 2018 ). However, evidence exists that some antisense transcripts are translated ( Ardern et al., 2020 ; Friedman et al., 2017 ; Stringer et al., 2021 ; Weaver et al., 2019 ). Our results ( Figures 2 A and 2B, second track) strongly support translation in both directions for the loci of PA1383 and tle3 .…”