The STAR protein Sam68 marks the transcriptionally active stages of spermatogenesis and regulates alternative splicing in male germ cells

Sam68 is an RNA binding protein belonging to the Signal Transduction and Activation of RNA (STAR) family. Sam68 is implicated in a variety of cellular processes including alternative splicing, nuclear export and cytoplasmic utilization of target mRNAs, and its function regulates cell cycle progression, cell survival and tumorigenesis. Sam68 is highly expressed in meiotic and post-meiotic male germ cells and Sam68 knockout male mice are infertile. We have previously demonstrated that Sam68 localizes in the cytoplasm and associates with the translation initiation complex eIF4F in secondary spermatocytes undergoing the meiotic divisions, and that Sam68 is required for translation of a subset of mRNAs during spermatogenesis. Nevertheless, Sam68 localization is mainly nuclear in pre-meiotic spermatocytes and post-meiotic spermatids, suggesting that nuclear functions of the protein are also relevant for spermatogenesis. In this study, we have analysed in detail the timing of expression of Sam68 during spermatogenesis, its interaction with the transcriptional machinery and its requirement for the regulation of alternative splicing of selected target pre-mRNAs. Using several approaches we demonstrate that expression of Sam68 is confined to the transcriptionally active stages of mouse spermatogenesis. By staining nuclear spreads with antibodies directed against Sam68 and SCP3, a component of the synaptonemal complex that holds together the homologous chromosomes in meiosis, we found that Sam68 expression is repressed at the onset of the first meiotic prophase (leptotene, zygotene and early pachytene spermatocytes) and it accumulates again at mid-pachytene and diplotene stages. Sam68 expression correlated with phosphorylation of the RNA polymerase II in serine 2, which is a hallmark of active transcription. Moreover, in mid-pachytene spermatocytes, Sam68 is excluded from the transcriptionally repressed sex body, indicating that Sam68 expression correlates temporally and physically with the transcriptionally active chromatin during germ cell differentiation. Moreover, we found that nuclear localization of Sam68 in permeabilized spermatocyte nuclei was lost upon treatment with RNAse or DNAse, suggesting that interaction with nucleic acids was required for its permanence in the nucleus. Given its nuclear localization and its well-recognized role in alternative splicing in many somatic cell types, we set out to analyze the role of Sam68 in the regulation of alternative pre-mRNA splicing in mouse germ cells. Using wild type and knockout purified germ cells, we identified a set of target mRNAs whose splicing is regulated by Sam68 in meiotic and post-meiotic germ cells. These results strongly suggest that Sam68 function in germ cells is intimately connected with its RNA binding activity and that its expression is required for the correct expression of specific mRNA isoforms during spermatogenesis.