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Recent proteogenomic approaches have led to the discovery that regions of the transcriptome previously annotated as non-coding regions [i.e., untranslated regions (UTRs), open reading frames overlapping annotated coding sequences in a different reading frame, and non-coding RNAs] frequently encode proteins, termed alternative proteins (altProts). This suggests that previously identified protein-protein interaction (PPI) networks are partially incomplete because altProts are not present in conventional protein databases. Here, we used the proteogenomic resource OpenProt and a combined spectrum- and peptide-centric analysis for the re-analysis of a high-throughput human network proteomics dataset, thereby revealing the presence of 261 altProts in the network. We found 19 genes encoding both an annotated (reference) and an alternative protein interacting with each other. Of the 117 altProts encoded by pseudogenes, 38 are direct interactors of reference proteins encoded by their respective parental genes. Finally, we experimentally validate several interactions involving altProts. These data improve the blueprints of the human PPI network and suggest functional roles for hundreds of altProts., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Pseudogenes are mutated copies of protein-coding genes that cannot be translated into proteins, but a small subset of pseudogenes has been detected at the protein level. Although ubiquitin pseudogenes represent one of the most abundant pseudogene families in many organisms, little is known about their expression and signaling potential. By re-analyzing public RNA-sequencing and proteomics datasets, we here provide evidence for the expression of several ubiquitin pseudogenes including UBB pseudogene 4 (UBBP4), which encodes Ub KEKS (Q2K, K33E, Q49K, N60S). The functional consequences of Ub KEKS conjugation appear to differ from canonical ubiquitylation. Quantitative proteomics shows that Ub KEKS modifies specific proteins including lamins. Knockout of UBBP4 results in slower cell division, and accumulation of lamin A within the nucleolus. Our work suggests that a subset of proteins reported as ubiquitin targets may instead be modified by ubiquitin variants that are the products of wrongly annotated pseudogenes and induce different functional effects.

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.

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.