Xavier Roucou

Although ribosome profiling (Ribo-seq) has revealed widespread translation of non-canonical open reading frames (ncORFs), integrating this information into routine mass spectrometry (MS) workflows remains challenging due to biochemical constraints and large search databases. We retrained TIS Transformer, a transcriptomic language model, by incorporating 40,397 Ribo-seq-derived non-canonical translation initiation sites, thereby enabling the prediction of 48,265 ncORFs, including non-AUG starts, with only a minor impact on canonical protein detection. We combined these predictions with Swiss-Prot sequences to create Swiss-Prot/ncProt, a size-controlled database suitable for standard proteomic analysis. Reanalysis of three independent MS datasets using this database maintained over 98% concordance with Swiss-Prot searches, identifying 541 non-canonical proteins, 429 of which had orthogonal evidence of their existence. This unified framework facilitates systematic detection of non-canonical proteins in conventional proteomics workflows, bridging the gap between canonical and non-canonical proteome analyses without requiring specialized experimental protocols.

A major scientific drive is to characterize the protein-coding genome, which is a primary basis for studying human health. But the fundamental question remains of what has been missed in previous analyses. Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states^1,2,3, with major implications for biomedical science. However, a key gap in knowledge has been which ncORFs produce small microproteins or alternative protein molecules that contribute to the human proteome. Here we report the collaborative efforts of the TransCODE Consortium⁴ to produce a consensus landscape of protein-level evidence for ncORFs. We show that about 25% of a set of 7,264 ncORFs gives rise to detectable peptides in a large-scale analysis of 95,520 proteomics experiments. We develop an annotation framework for ncORF-encoded microproteins as human proteins and codify the new conceptual model of ‘peptideins’ as microproteins that have indeterminate potential as functional proteins. To probe the biological implications of peptideins, we create an evolutionary analysis approach, termed ORF relative branch length (ORBL), and determine that evolutionary constraint is common and associates with observation of ncORF-derived peptides. We then characterize a pan-essential cellular phenotype for one peptidein from the OLMALINC long non-coding RNA. Overall, we generate public research tools supported by GENCODE and PeptideAtlas and advance biomedical discovery for understudied components of the human proteome.

Non-canonical (i.e. unannotated) open reading frames (ncORFs) have until recently been omitted from reference genome annotations, despite evidence of their translation, limiting their incorporation into biomedical research. To address this, in 2022, we initiated the TransCODE consortium and built the first community-driven consensus catalog of human ncORFs, which was openly distributed to the research community via Ensembl-GENCODE. While this catalog represented a starting point for reference ncORF annotation, major technical and scientific issues remained. In particular, this initial catalog had no standardized framework to judge the evidence of translation for individual ncORFs. Here, we present an expanded and refined catalog of the human reference annotation of ncORFs. By incorporating more datasets and by lifting constraints on ORF length and start codon, we define a comprehensive set of 28 359 ncORFs that is nearly four times the size of the previous catalog. Furthermore, to aid users who wish to work with ncORFs with the strongest and most reproducible signals of translation, we utilized a data-driven framework (i.e. translation signature scores) to assess the accumulated evidence for any individual ncORF. Using this approach, we derive a subset of 10 127 ncORFs with translation evidence on par with canonical protein-coding genes, which we refer to as the primary set. This set can serve as a reliable reference for downstream analyses and validation, with a particular emphasis on high quality. Overall, this update reflects continuous community-driven efforts to make ncORFs accessible and actionable to the broader research public, and further iterations of the catalog will continue to expand and refine this resource.

SUMMARY: Non-canonical open reading frames (ncORFs) are an emerging area of research that is quickly gaining momentum. Many peptides and proteins missed in initial annotation efforts (ncProts) were subsequently shown to be crucial for a wide range of biological processes. The discovery of ncORFs continues to improve the accuracy of loss-of-function studies because they often occupy the same genomic spaces as annotated ORFs. While databases of mutant phenotypes linked to genomic loci exist in a few species, none of these databases integrate the information on ncORFs present in already characterized loci. In this study, we introduce a nearly comprehensive loss-of-function phenomics dataset of Medicago truncatula (673 loci characterized over the past 30 years), which was integrated as a new track into the genome browser of this organism. This dataset helped critically analyze the potential contribution of ncORFs to published phenotypes. We detected mass spectrometry (MS)-validated ncORFs in 10 characterized genes, including major regulators of development and symbiotic relationships. We also found conserved ncORFs in 113 characterized genes, including four genes with highly conserved ncORFs. In some studies, the contribution of these ncORFs can be ruled out, while in others it cannot. Using real examples, we systematized ambiguities associated with ncORFs. Furthermore, we highlighted little-known trans effects of insertional mutagenesis on splicing as contributors to that ambiguity. Finally, our meta-analysis of published phenotypes revealed that different protein classes have significantly different (unique) proportions of unconditional, conditional, and neutral phenotypes, potentially reflecting their relative functional importance. Significance statement: This study is the first to merge a nearly comprehensive inventory of loss-of-function studies in a eukaryotic organism with the information on novel MS-validated and conserved ncORFs.

Thousands of short open reading frames (sORFs) are translated outside of annotated coding sequences. Recent studies have pioneered searching for sORF-encoded microproteins in mass spectrometry (MS)-based proteomics and peptidomics datasets. Here, we assessed literature-reported MS-based identifications of unannotated human proteins. We find that studies vary by three orders of magnitude in the number of unannotated proteins they report. Of nearly 10,000 reported sORF-encoded peptides, 96% were unique to a single study, and 12% mapped to annotated proteins or proteoforms. Manual curation of a benchmark dataset of 406 manually evaluated spectra from 204 sORF-encoded proteins revealed large variation in peptide-spectrum match (PSM) quality between studies, with immunopeptidomics studies generally reporting higher quality PSMs than conventional enzymatic digests of whole cell lysates. We estimate that 65% of predicted sORF-encoded protein detections in immunopeptidomics studies were supported by high-quality PSMs versus 7.8% in non-immunopeptidomics datasets. Our work stresses the need for standardized protocols and analysis workflows to guide future advancements in microprotein detection by MS towards uncovering how many human microproteins exist.

Non-canonical (i.e., unannotated) open reading frames (ncORFs) have until recently been omitted from reference genome annotations, despite evidence of their translation, limiting their incorporation into biomedical research. To address this, in 2022, we initiated the TransCODE consortium and built the first community-driven consensus catalog of human ncORFs, which was openly distributed to the research community via Ensembl-GENCODE. While this catalog represented a starting point for reference ncORF annotation, major technical and scientific issues remained. In particular, this initial catalogue had no standardized framework to judge the evidence of translation for individual ncORFs. Here, we present an expanded and refined catalog of the human reference annotation of ncORFs. By incorporating more datasets and by lifting constraints on ORF length and start-codon, we define a comprehensive set of 28,359 ncORFs that is nearly four times the size of the previous catalog. Furthermore, to aid users who wish to work with ncORFs with the strongest and most reproducible signals of translation, we utilized a data-driven framework (i.e. translation signature scores) to assess the accumulated evidence for any individual ncORF. Using this approach, we derive a subset of 7,888 ncORFs with translation evidence on par with canonical protein-coding genes, which we refer to as the Primary set. This set can serve as a reliable reference for downstream analyses and validation, with a particular emphasis on high quality. Overall, this update reflects continual community-driven efforts to make ncORFs accessible and actionable to the broader research public and further iterations of the catalog will continue to expand and refine this resource.

Proliferating Cell Nuclear Antigen (PCNA) is a pivotal regulator of DNA replication and repair, orchestrating protein recruitment through its binding domains and post-translational modifications (PTMs). Lysine 164 (K164) of PCNA serves as a critical molecular switch modulated by ubiquitin (Ub) and ubiquitin-like proteins. Here, we identify the ubiquitin variant Ub^KEKS as a new PCNA regulator, demonstrating its covalent modification of K164 and quantifying the Ub^KEKS-to-Ub modification ratio. Loss of Ub^KEKS results in delayed S-phase progression without altering PCNA levels or nuclear localization. However, DNA fiber combing assays reveal a significant reduction in DNA polymerase processivity in Ub^KEKS-deficient cells, indicating its role in replication efficiency. At a whole-cell scale, the mapping by mass spectrometry of diGly remnants after trypsin digestion, demonstrate that modifications by Ub^KEKS or Ub do not compensate one another, due to key differences regarding amino acids surrounding modified lysines. Ultimately, our findings establish Ub^KEKS as a distinct key modulator of PCNA function, expanding the repertoire of PTMs that influence DNA replication dynamics. These insights pave the way for further exploration of Ub^KEKS as a regulator of genome stability and cell cycle regulation.

Thousands of short open reading frames (sORFs) are translated outside of annotated coding sequences. Recent studies have pioneered searching for sORF-encoded microproteins in mass spectrometry (MS)- based proteomics and peptidomics datasets. Here, we assessed literature-reported MS-based identifications of unannotated human proteins. We find that studies vary by three orders of magnitude in the number of unannotated proteins they report. Of nearly 10,000 reported sORF-encoded peptides, 96% were unique to a single study, and 12% mapped to annotated proteins or proteoforms. Manual curation of a benchmark dataset of 406 manually evaluated spectra from 204 sORF-encoded proteins revealed large variation in peptide-spectrum match (PSM) quality between studies, with immunopeptidomics studies generally reporting higher quality PSMs than conventional enzymatic digests of whole cell lysates. We estimate that 65% of predicted sORF-encoded protein detections in immunopeptidomics studies were supported by high-quality PSMs versus 7.8% in non-immunopeptidomics datasets. Our work stresses the need for standardized protocols and analysis workflows to guide future advancements in microprotein detection by MS towards uncovering how many human microproteins exist.