Small Open Reading Frames, How to Find Them and Determine Their Function

Abstract

Advances in genomics and molecular biology have revealed an abundance of small open reading frames (sORFs) across all types of transcripts. While these sORFs are often assumed to be non-functional, many have been implicated in physiological functions and a significant number of sORFs have been described in human diseases. Thus, sORFs may represent a hidden repository of functional elements that could serve as therapeutic targets. Unlike protein-coding genes, it is not necessarily the encoded peptide of an sORF that enacts its function, sometimes simply the act of translating an sORF might have a regulatory role. Indeed, the most studied sORFs are located in the 5′UTRs of coding transcripts and can have a regulatory impact on the translation of the downstream protein-coding sequence. However, sORFs have also been abundantly identified in non-coding RNAs including lncRNAs, circular RNAs and ribosomal RNAs suggesting that sORFs may be diverse in function. Of the many different experimental methods used to discover sORFs, the most commonly used are ribosome profiling and mass spectrometry. These can confirm interactions between transcripts and ribosomes and the production of a peptide, respectively. Extensions to ribosome profiling, which also capture scanning ribosomes, have further made it possible to see how sORFs impact the translation initiation of mRNAs. While high-throughput techniques have made the identification of sORFs less difficult, defining their function, if any, is typically more challenging. Together, the abundance and potential function of many of these sORFs argues for the necessity of including sORFs in gene annotations and systematically characterizing these to understand their potential functional roles. In this review, we will focus on the high-throughput methods used in the detection and characterization of sORFs and discuss techniques for validation and functional characterization.

Common and Rare 5′UTR Variants Altering Upstream Open Reading Frames in Cardiovascular Genomics

“…We did not discuss molecular tools that are available to determine whether these upORFs could be at the origin of functional small micropeptides that could have specific physiological roles. This topic has recently been addressed in an independent review ( 28 ). Finally, we only examined in this work SNVs that could create uAUG resulting upORFs, the most known class of variants among those that affect non-canonical ORFs.…”

“…These assays are based on the cloning of the entire promoter of a given transcript before the coding sequence of a luciferase and the evaluation of the promoter activity in wild-type and mutant contexts in vitro by measuring the obtained luciferase luminescence normalized to a control vector. Additional methods used to characterize small ORFs and their potential translation into SEPs has been recently reviewed in ( 28 ). Altogether, upSNVs are still a neglected class of non-coding variations, and are often called as Variants of Unknown Significance when they are identified in routine clinical diagnosis, contributing then to medical wandering.…”

Kidney diseases and long non-coding RNAs in the limelight

“…(Derrien et al, 2012;Alessio et al, 2020). Previous studies have reported that lncRNAs are unable to encode proteins, but recent investigations have identified abundant small open reading frames in lncRNAs via ribosomal analysis, some of which have the potential to encode micro-peptides and proteins (Kong et al, 2020;Ye et al, 2020;Zhou B. et al, 2021;Kute et al, 2021). RNA polymerase II (Pol II) transcribes protein-encoding genes and numerous pre-messenger RNAs (mRNAs) of lncRNAs, including long intergenic non-coding RNAs (lincRNAs), enhancer RNAs, antisense RNAs, and promoter upstream transcripts (Nojima and Proudfoot, 2022).…”

“…( Derrien et al, 2012 ; Alessio et al, 2020 ). Previous studies have reported that lncRNAs are unable to encode proteins, but recent investigations have identified abundant small open reading frames in lncRNAs via ribosomal analysis, some of which have the potential to encode micro-peptides and proteins ( Kong et al, 2020 ; Ye et al, 2020 ; Zhou B. et al, 2021 ; Kute et al, 2021 ).…”

In silicocomparative RNA-seq analysis reveals varietal-specific intergenic small open reading frames inCucumis sativusL.

“…Their minuscule size has caused them to be excluded from most gene prediction methods (Couso and Patraquim, 2017). The length cut-off filter used in most gene prediction methods is 300 bp and any sequences below this cut-off will be considered as being non-functional (Kute et al, 2022).…”