title: Forty-four novel protein-coding loci discovered using a proteomics informed by transcriptomics (PIT) approach in rat male germ cells.
abstract: Spermatogenesis is a complex process, dependent upon the successive activation and/or repression of thousands of gene products, and ends with the production of haploid male gametes. RNA sequencing of male germ cells in the rat identified thousands of novel testicular unannotated transcripts (TUTs). Although such RNAs are usually annotated as long noncoding RNAs (lncRNAs), it is possible that some of these TUTs code for protein. To test this possibility, we used a "proteomics informed by transcriptomics" (PIT) strategy combining RNA sequencing data with shotgun proteomics analyses of spermatocytes and spermatids in the rat. Among 3559 TUTs and 506 lncRNAs found in meiotic and postmeiotic germ cells, 44 encoded at least one peptide. We showed that these novel high-confidence protein-coding loci exhibit several genomic features intermediate between those of lncRNAs and mRNAs. We experimentally validated the testicular expression pattern of two of these novel protein-coding gene candidates, both highly conserved in mammals: one for a vesicle-associated membrane protein we named VAMP-9, and the other for an enolase domain-containing protein. This study confirms the potential of PIT approaches for the discovery of protein-coding transcripts initially thought to be untranslated or unknown transcripts. Our results contribute to the understanding of spermatogenesis by characterizing two novel proteins, implicated by their strong expression in germ cells. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium under the data set identifier PXD000872.
authors.affiliation: Proteomics Core Facility Biogenouest, Inserm U1085, IRSET, Campus de Beaulieu, Rennes, France Inserm U1085, IRSET, Université de Rennes 1, Rennes, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Chocu
authors.affiliation: Inserm U1085, IRSET, Université de Rennes 1, Rennes, France.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Evrard
authors.affiliation: Proteomics Core Facility Biogenouest, Inserm U1085, IRSET, Campus de Beaulieu, Rennes, France Inserm U1085, IRSET, Université de Rennes 1, Rennes, France.
authors.forename: Régis
authors.initials: R
authors.lastname: Lavigne
authors.affiliation: Inserm U1085, IRSET, Université de Rennes 1, Rennes, France.
authors.forename: Antoine D
authors.initials: AD
authors.lastname: Rolland
authors.affiliation: Inserm U1085, IRSET, Université de Rennes 1, Rennes, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Aubry
authors.affiliation: Inserm U1085, IRSET, Université de Rennes 1, Rennes, France.
authors.forename: Bernard
authors.initials: B
authors.lastname: Jégou
authors.affiliation: Inserm U1085, IRSET, Université de Rennes 1, Rennes, France frederic.chalmel@inserm.fr.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Chalmel
authors.affiliation: Proteomics Core Facility Biogenouest, Inserm U1085, IRSET, Campus de Beaulieu, Rennes, France Inserm U1085, IRSET, Université de Rennes 1, Rennes, France charles.pineau@inserm.fr.
authors.forename: Charles
authors.initials: C
authors.lastname: Pineau
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D019295:Computational Biology; D020869:Gene Expression Profiling / Q000379:methods*; D050437:Genes, Developmental; D056726:Genetic Association Studies / Q000379:methods*; D056426:Genetic Loci; D008297:Male; D016366:Open Reading Frames / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013093:Spermatogonia / Q000378:metabolism*; D059467:Transcriptome
keywords: RNA profiling; proteomics; spermatogenesis; testicular unannotated transcripts; transcriptome