Revisiting sORFs: overcoming challenges to identify and characterize functional microproteins

Abstract

Short ORFs (sORFs), that is, occurrences of a start and stop codon within 100 codons or less, can be found in organisms of all domains of life, outnumbering annotated protein-coding ORFs by orders of magnitude. Even though functional proteins smaller than 100 amino acids are known, the coding potential of sORFs has often been overlooked, as it is not trivial to predict and test for functionality within the large number of sORFs. Recent advances in ribosome profiling and mass spectrometry approaches, together with refined bioinformatic predictions, have enabled a huge leap forward in this field and identified thousands of likely coding sORFs. A relatively low number of small proteins or microproteins produced from these sORFs have been characterized so far on the molecular, structural, and/or mechanistic level. These however display versatile and, in some cases, essential cellular functions, allowing for the exciting possibility that many more, previously unknown small proteins might be encoded in the genome, waiting to be discovered. This review will give an overview of the steadily growing microprotein field, focusing on eukaryotic small proteins. We will discuss emerging themes in the molecular action of microproteins, as well as advances and challenges in microprotein identification and characterization. Abbreviations dN/dS, nonsynonymous versus synonymous mutation ratio; dORF, downstream open reading frame; IDP, intrinsically disordered protein; lncRNA, long noncoding RNA; MHC I, major histocompatibility complex class I; MS, mass spectrometry; ORF, open reading frame; Riboseq, Ribosome sequencing/profiling; SEP, short open reading frame-encoded polypeptide; sORF or smORF, short open reading frame; uORF, upstream open reading frame.

A vast evolutionarily transient translatome contributes to phenotype and fitness

“…Indeed, research on microproteins show clearly that sequences shorter than 100 codons are often biologically important. 5,50 It is perhaps surprising that a coding sequence can affect organism phenotype despite showing no evidence of selection. However, this result is consistent with evidence from the field of de novo gene birth.…”

Overlapping genes in natural and engineered genomes

“…Furthermore, it is likely that many of the ncRNAs found to possess sORFs are in fact misannotated and should be re-defined as mRNA; however, there are examples of RNA that possess dual functionality (non-coding and coding), thereby complicating classifications 118 , 119 . More information on this developing area can be found in recent reviews 120 , 121 , including the in-motion and recent community-driven initiative to comprehensively define and catalogue these classes of non-canonical ORFs in major databases 14 .…”

Bottom‐up and top‐down proteomic approaches for the identification, characterization, and quantification of the low molecular weight proteome with focus on short open reading frame‐encoded peptides

“…Meanwhile, the functions of some SEPs in, for example, cell division, membrane transport, embryonic development, or cell death have been elucidated. Different biological functions of SEPs have been reviewed [12,15,93,94].…”