Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

Abstract

A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.

Translating DRiPs: MHC class I immunosurveillance of pathogens and tumors

“…The abundance-alternative ORFs were corroborated [161], supporting the idea that alternative ORFs provide unexpected complexity for immunosurveillance to exploit. Some peptides identified derive from initiation at a near-cognate non-AUG codon [159,160].…”

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

“…Another study, identified 1,259 novel peptides [46], by matching the spectra of different HPLC-MS data-sets (16 in total, covering a range of different human samples) to a custom database of predicted ORFs within annotated transcripts (mapping to UTRs and overlapping CDS'), suggesting that the translation of these "alternative" smORFs could be a wide-spread phenomenon. Interestingly, the majority of these peptides were identified in plasma and serum samples (1,118 / 1,259), implying that they could be secreted, although the reason or mechanism leading to this remains unknown (Table S1).…”

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

“…Phylogenetic evidence also strongly supports the functional importance of this uORF product in addition to the uORF's regulatory role in providing stress resistance to the translation of the annotated CDS 45. The detection of a product translated from an ORF in the 3′UTR of the human CHTF8 gene has also been reported 43, 44. Indeed in GWIPS‐viz, a high ribosome occupancy can be seen in the 3′UTR region of the human CHTF8 transcripts (Fig.…”