Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation

Abstract

Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo.

Global Analysis of Truncated RNA Ends Reveals New Insights into Ribosome Stalling in Plants

“…We predicted that, if PARE can capture ribosome footprints in the same way as ribosome profiling (Ribo-Seq), which delineates ribosome positions by generating ribosome-protected mRNA fragments through in vitro nuclease digestion (Ingolia, 2010), we would observe a 3-nucleotide periodicity in the CDSs. A 3-nucleotide periodicity reflects the stepwise movement of ribosomes during active translation and has been reported in the analyses of Ribo-Seq data derived from multiple species (Ingolia et al, 2009(Ingolia et al, , 2011Guo et al, 2010;Liu et al, 2013;Bazzini et al, 2014;Juntawong et al, 2014;Vasquez et al, 2014). Consistent with our prediction, 59 ends of truncated mRNA (PARE data) generated from Arabidopsis seedlings and inflorescences show strong 3-nucleotide phasing in the 39 terminus of the CDSs but not in the proximal region of the 39 UTR ( Figure 1A).…”

“…Although the enrichment in the translational frame is relatively small in PARE data compared with that in yeast Ribo-Seq data reported previously (Ingolia et al, 2009), the proportion of PARE reads falling in the translational frame is significantly higher than that in the other two frames in the three replicates of Arabidopsis inflorescence PARE data (Supplemental Figure 1). Similar to the previous finding in the analysis of Ribo-Seq data (Liu et al, 2013;Bazzini et al, 2014;Juntawong et al, 2014), PARE data of these three plant species also showed an evident increase in the number of reads at positions 16 and 17 nucleotides upstream of stop codons, a pattern consistent with the deceleration of ribosome movement during translational termination ( Figure 1A). These common features shared between PARE data and Ribo-Seq data strongly suggest both the presence of in vivo ribosome-protected mRNA fragments in the plant RNA degradome and the occurrence of cotranslational RNA decay in plants.…”

Mixed messages: Re-initiation factors regulate translation of animal mRNAs

“…Intriguingly, using methods that we originally applied in vertebrates [10], we noticed that several uORFs with DENR-dependent effects on translation are conserved in this sense. Some reside on mRNAs encoding important transcription factors like cryptocephal or gemini.…”

“…In some cases, a uORFencoded small peptide can interfere with translation in cis or in trans [3]. In fact, many small ORFs in the 5′ or 3′ UTRs of mRNAs are not only translated but also give rise to detectable small peptides [9,10]. Some even show clear evolutionary signatures of negative selection on the encoded amino acid sequence, suggesting functionality of the peptide product.…”

GWIPS‐viz as a tool for exploring ribosome profiling evidence supporting the synthesis of alternative proteoforms

“…The answer to this question may be determined with subsequent analysis of the triplet periodicity within the corresponding region 41, 42. However, even in the absence of periodicity analysis, a simple examination of the ORF organization could rule out the possibility of an extended CDS.…”