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

  • ️Sat Jan 01 2022

Review

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

Preeti Madhav Kute et al. Front Genet. 2022.

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.

Keywords: SEPs; computational tools; mass spectrometry; ribosome profiling; sORFs.

Copyright © 2022 Kute, Soukarieh, Tjeldnes, Trégouët and Valen.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1

Examples of small ORFs in coding (A) and non-coding (B) transcripts. Start and Stop indicate the initiation and termination sites of the coding sequence (CDS). uORF, upstream open reading frame fully located in the 5′UTR; uStart, upstream start site; uStop, upstream stop site; uoORF, upstream overlapping open reading frame; intStart, internal start site; intORF, internal open reading frame; intStop, internal stop site; dStart, downstream Start site; dStop, downstream stop site; sORF, small open reading frame; lncRNA, long non-coding RNA; circRNA, circular RNA.

FIGURE 2
FIGURE 2

Overview of the commonly used techniques to identify and characterize sORFs and their encoded peptides. Novel sORFs and their products can be detected by the prediction algorithms using bioinformatic approaches, by generating peptide databases using improved mass spectrometry-based assays and by using ribosome profiling and related sequencing techniques to obtain translationally active transcripts. The predicted SEPs can be validated by various assays such as reporter-based overexpression, epitope tagging etc. Loss of function assays could be done to assess the cellular function of these SEPs.

FIGURE 3
FIGURE 3

Profiling and sequencing of translating transcripts. A254 profiles shown before (A) and after digestion with ribonucleases (B,C). The fractions used for further processing are highlighted, polysomes in purple, 80S in orange and 40S in green. (D) The process of library preparation for next generation sequencing. Size selection of ∼30 nt is done for ribosome profiling and ribosome complex profiling sequencing and libraries are prepared from the size selected small RNAs, whereas for polysome profiling, libraries are prepared from total RNA. Meta-coverage shown for reads obtained from polysome profiling sequencing (E), for ribosome profiling (F) and for ribosome complex profiling [(G) top: 40S, bottom: 80S].

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