Discovery of Human sORF-Encoded Polypeptides (SEPs) in Cell Lines and Tissue

Abstract

The existence of nonannotated protein-coding human short open reading frames (sORFs) has been revealed through the direct detection of their sORF-encoded polypeptide (SEP) products. The discovery of novel SEPs increases the size of the genome and the proteome and provides insights into the molecular biology of mammalian cells, such as the prevalent usage of non-AUG start codons. Through modifications of the existing SEP-discovery workflow, we discover an additional 195 SEPs in K562 cells and extend this methodology to identify novel human SEPs in additional cell lines and human tissue for a final tally of 237 new SEPs. These results continue to expand the human genome and proteome and demonstrate that SEPs are a ubiquitous class of nonannotated polypeptides that require further investigation.

A Human Short Open Reading Frame (sORF)-encoded Polypeptide That Stimulates DNA End Joining

“…1A) (6,7). Because this SEP was annotated as a predicted protein in the NCBI nonredundant protein database, we did not include it in the list of reported SEPs (7); however, our evidence is the first experimental detection of this polypeptide in cells.…”

“…In particular, widespread translation of short open reading frames (1) (sORFs) 3 producing peptides Ͻ150 amino acids has been reported in mammalian cells by ribosome profiling (2,3), proteomics (4), and peptidomics (5)(6)(7)(8). The apparent prevalence of sORF translation raises the question of whether sORF-encoded polypeptides (SEPs) are functional biomolecules.…”

Recognition of the polycistronic nature of human genes is critical to understanding the genotype-phenotype relationship

“…Yet, it is clear it also leads to numerous false negatives, i.e., functional ORFs shorter than 100 codons excluded from annotations (Andrews and Rothnagel 2014;Ma et al 2014;Pauli et al 2014;Couso and Patraquim 2017). Notwithstanding, we could also question the arbitrary cut-off taken by groups studying alternative ORFs.…”

“…These criteria result in an important underestimation of translated ORFs in the genome (Andrews and Rothnagel 2014;Saghatelian and Couso 2015;Couso and Patraquim 2017;Plaza et al 2017). With functional evidence for previously unannotated ORFs in bacteria (Wadler and Vanderpool 2007;Hemm et al 2008Hemm et al , 2010Storz et al 2014;Lluch-Senar et al 2015;Baek et al 2017), Drosophila (Galindo et al 2007;Kondo et al 2007;Reinhardt et al 2013;Aspden et al 2014;Albuquerque et al 2015;Li et al 2016a;Pueyo et al 2016a), plants (Hanada et al 2013;Juntawong et al 2014;Hsu et al 2016;Hsu and Benfey 2017), and other eukaryotes (Oyama et al 2007;Ingolia et al 2011;Vanderperre et al 2013;Ma et al 2014), genome annotations will need to be revised.…”

New Peptides Under the s(ORF)ace of the Genome

“…This method was tested against other workflows [45], leading to two important observations: first, the use of ERLIC fractionation greatly increases the number of peptides detected (~10 fold) and second, there is an important lack of overlap between the peptides identified by different workflows, and even by different technical repeats, highlighting the stochastic nature of this technique, and the requirement of several repeats to achieve an optimal sampling saturation of the peptidome. In total, they analysed 3 different cell lines and a tumor sample, and identified a total of 311 short peptides, of which 237 are novel, with ~80% of them mapping to previously unannotated transcripts (Table 1), and the rest to alternative CDS within annotated transcripts with a similar distribution, in UTRs and overlapping CDS', as found by Slavoff et al [44].…”