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.

MRNAs carry the genetic information that is translated by ribosomes. The traditional view of a mature eukaryotic mRNA is a molecule with three main regions, the 5′ UTR, the protein coding open reading frame (ORF) or coding sequence (CDS), and the 3′ UTR. This concept assumes that ribosomes translate one ORF only, generally the longest one, and produce one protein. As a result, in the early days of genomics and bioinformatics, one CDS was associated with each protein-coding gene. This fundamental concept of a single CDS is being challenged by increasing experimental evidence indicating that annotated proteins are not the only proteins translated from mRNAs. In particular, mass spectrometry (MS)-based proteomics and ribosome profiling have detected productive translation of alternative open reading frames. In several cases, the alternative and annotated proteins interact. Thus, the expression of two or more proteins translated from the same mRNA may offer a mechanism to ensure the co-expression of proteins which have functional interactions. Translational mechanisms already described in eukaryotic cells indicate that the cellular machinery is able to translate different CDSs from a single viral or cellular mRNA. In addition to summarizing data showing that the protein coding potential of eukaryotic mRNAs has been underestimated, this review aims to challenge the single translated CDS dogma.

OpenProt (www.openprot.org) is the first proteogenomic resource supporting a polycistronic annotation model for eukaryotic genomes. It provides a deeper annotation of open reading frames (ORFs) while mining experimental data for supporting evidence using cutting-edge algorithms. This update presents the major improvements since the initial release of OpenProt. All species support recent NCBI RefSeq and Ensembl annotations, with changes in annotations being reported in OpenProt. Using the 131 ribosome profiling datasets re-analysed by OpenProt to date, non-AUG initiation starts are reported alongside a confidence score of the initiating codon. From the 177 mass spectrometry datasets re-analysed by OpenProt to date, the unicity of the detected peptides is controlled at each implementation. Furthermore, to guide the users, detectability statistics and protein relationships (isoforms) are now reported for each protein. Finally, to foster access to deeper ORF annotation independently of one’s bioinformatics skills or computational resources, OpenProt now offers a data analysis platform. Users can submit their dataset for analysis and receive the results from the analysis by OpenProt. All data on OpenProt are freely available and downloadable for each species, the release-based format ensuring a continuous access to the data. Thus, OpenProt enables a more comprehensive annotation of eukaryotic genomes and fosters functional proteomic discoveries.

Advances in proteomics and sequencing have highlighted many non-annotated open reading frames (ORFs) in eukaryotic genomes. Genome annotations, cornerstones of today's research, mostly rely on protein prior knowledge and on ab initio prediction algorithms. Such algorithms notably enforce an arbitrary criterion of one coding sequence (CDS) per transcript, leading to a substantial underestimation of the coding potential of eukaryotes. Here, we present OpenProt, the first database fully endorsing a polycistronic model of eukaryotic genomes to date. OpenProt contains all possible ORFs longer than 30 codons across 10 species, and cumulates supporting evidence such as protein conservation, translation and expression. OpenProt annotates all known proteins (RefProts), novel predicted isoforms (Isoforms) and novel predicted proteins from alternative ORFs (AltProts). It incorporates cutting-edge algorithms to evaluate protein orthology and re-interrogate publicly available ribosome profiling and mass spectrometry datasets, supporting the annotation of thousands of predicted ORFs. The constantly growing database currently cumulates evidence from 87 ribosome profiling and 114 mass spectrometry studies from several species, tissues and cell lines. All data is freely available and downloadable from a web platform (www.openprot.org) supporting a genome browser and advanced queries for each species. Thus, OpenProt enables a more comprehensive landscape of eukaryotic genomes’ coding potential.