Forty-four novel protein-coding loci discovered using a PIT approach in rat male germ cells

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, named TUTs. Although such RNAs are usually annotated as long non-coding RNAs, it is possible that some of these TUTs code for protein. To test this possibility, we used a “Proteomics Informed by Transcriptomics” strategy, combining shotgun proteomics analyses and RNA sequencing data for enriched populations of rat testicular cells. Among 3559 TUTs and 506 lncRNAs found in meiotic and post-meiotic germ cells, 44 encoded at least one peptide. We show 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.