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.

Recent functional, proteomic and ribosome profiling studies in eukaryotes have concurrently demonstrated the translation of alternative open-reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). We show that a large number of small proteins could in fact be coded by these altORFs. The putative alternative proteins translated from altORFs have orthologs in many species and contain functional domains. Evolutionary analyses indicate that altORFs often show more extreme conservation patterns than their CDSs. Thousands of alternative proteins are detected in proteomic datasets by reanalysis using a database containing predicted alternative proteins. This is illustrated with specific examples, including altMiD51, a 70 amino acid mitochondrial fission-promoting protein encoded in MiD51/Mief1/SMCR7L, a gene encoding an annotated protein promoting mitochondrial fission. Our results suggest that many genes are multicoding genes and code for a large protein and one or several small proteins.

An endoproteolytic cleavage termed ␣-cleavage between residues 111/112 is a characteristic feature of the cellular prion protein (PrP C ). This cleavage generates a soluble N-terminal fragment (PrPN1) and a glycosylphosphatidylinositol-anchored C-terminal fragment (PrPC1). Independent studies demonstrate that modulating PrP C ␣-cleavage represents a potential therapeutic strategy in prion diseases. The regulation of PrP C ␣-cleavage is unclear. The only known domain that is essential for the ␣-cleavage to occur is a hydrophobic domain (HD). Importantly, the HD is also essential for the formation of PrP C homodimers. To explore the role of PrP C homodimerization on the ␣-cleavage, we used a well described inducible dimerization strategy whereby a chimeric PrP C composed of a modified FK506-binding protein (Fv) fused with PrP C and termed Fv-PrP is incubated in the presence of a dimerizer AP20187 ligand. We show that homodimerization leads to a considerable increase of PrP C ␣-cleavage in cultured cells and release of PrPN1 and PrPC1. Interestingly, enforced homodimerization increased PrP C levels at the plasma membrane, and preventing PrP C trafficking to the cell surface inhibited dimerization-induced ␣-cleavage. These observations were confirmed in primary hippocampal neurons from transgenic mice expressing Fv-PrP. The proteases responsible for the ␣-cleavage are still elusive, and in contrast to initial studies we confirm more recent investigations that neither ADAM10 nor ADAM17 are involved. Importantly, PrPN1 produced after PrP C homodimerization protects against toxic amyloid-␤ (A␤) oligomers. Thus, our results show that PrP C homodimerization is an important regulator of PrP C ␣-cleavage and may represent a potential therapeutic avenue against A␤ toxicity in Alzheimer's disease.

Background:The spinocerebellar ataxia type 1 (SCA1) gene encoding ataxin-1 (ATXN1) contains an alternative open reading frame overlapping the ATXN1 coding sequence. Results: The alternative ATXN1 protein is constitutively co-expressed and interacts with ATXN1 N terminus. Alternative ATXN1 is also an RNA binding protein. Conclusion:ATXN1 is a genuine dual coding gene. Significance: Alternative ATXN1 may regulate the function of ATXN1 in physiological and pathological conditions.