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.
Highlights
The proteome impacts all aspects of health and disease and deciphering the human proteome represents an important challenge in the post-genomic era
We defined alternative open reading frames (AltORFs) as ORFs located in a non-canonical reading frame of the reference ORFs (RefORFs), in the 59 and 39UTR regions of an mRNA, or partially overlapping with both the RefORF and an untranslated regions (UTRs) region (Fig. 1A)
Criteria included in previous predictions 2conservation among species, presence of an optimal Kozak context around the initiator AUG codon, location within the reference coding sequence2 were not taken into account in this study because experimental evidence indicate that these criteria are not necessarily required for an AltORF to be expressed [11,12]
Summary
The proteome impacts all aspects of health and disease and deciphering the human proteome represents an important challenge in the post-genomic era. A typical fully processed mRNA includes one RefORF and is associated with a reference protein (Fig. 1A). Reference proteins populate current protein databases used to support research in life sciences. Two cellular mechanisms have evolved to increase proteomic diversity by encoding more than one protein per gene, increasing the diversity of the transcriptome or producing more than one protein from a single transcript. Transcriptome diversity [2] is achieved by utilization of alternative promoters [3], reiterative transcription [4], or post-transcriptional processing, including alternative splicing [5], alternative polyadenylation [6] and RNA editing [7]. N-terminal extension [8], ribosomal frameshifting [9,10] and utilization of multiple coding ORFs in one transcript [11,12] can generate functional or diseaserelated proteins
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