SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma

Mammalian Pumilio (PUM) proteins are sequence-specific, RNA-binding proteins (RBPs) with wide-ranging roles. They are involved in germ cell development, which has functional implications in development and fertility. Although human PUM1 and PUM2 are closely related to each other and recognize the same RNA binding motif, there is some evidence for functional diversity. To address that problem, first we used RIP-Seq and RNA-Seq approaches, and identified mRNA pools regulated by PUM1 and PUM2 proteins in the TCam-2 cell line, a human male germ cell model. Second, applying global mass spectrometry-based profiling, we identified distinct PUM1- and PUM2-interacting putative protein cofactors, most of them involved in RNA processing. Third, combinatorial analysis of RIP and RNA-Seq, mass spectrometry, and RNA motif enrichment analysis revealed that PUM1 and PUM2 form partially varied RNP-regulatory networks (RNA regulons), which indicate different roles in human reproduction and testicular tumorigenesis. Altogether, this work proposes that protein paralogues with very similar and evolutionary highly conserved functional domains may play divergent roles in the cell by combining with different sets of protein cofactors. Our findings highlight the versatility of PUM paralogue-based post-transcriptional regulation, offering insight into the mechanisms underlying their diverse biological roles and diseases resulting from their dysfunction.

While two mouse NANOS paralogues, NANOS2 and NANOS3, are crucial for maintenance of germ cells by suppression of apoptosis, the mouse NANOS1 paralogue does not seem to regulate these processes. Previously, we described a human NANOS1 p.[(Pro34Thr);(Ser83del)] mutation associated with the absence of germ cells in seminiferous tubules of infertile patients, which might suggest an anti-apoptotic role of human NANOS1. In this study, we aimed to determine a potential influence of human NANOS1 on the maintenance of TCam-2 model germ cells by investigating proliferation, cell cycle, and apoptosis. Constructs encoding wild-type or mutated human NANOS1 were used for transfection of TCam-2 cells, in order to investigate the effect of NANOS1 on cell proliferation, which was studied using a colorimetric assay, as well as apoptosis and the cell cycle, which were measured by flow cytometry. RNA-Seq (RNA sequencing) analysis followed by RT-qPCR (reverse transcription and quantitative polymerase chain reaction) was conducted for identifying pro-apoptotic genes repressed by NANOS1. Here, we show that overexpression of NANOS1 downregulates apoptosis in TCam-2 cells. Moreover, we found that NANOS1 represses a set of pro-apoptotic genes at the mRNA level. We also found that the infertility-associated p.[(Pro34Thr);(Ser83del)] mutation causes NANOS1 to functionally switch from being anti-apoptotic to pro-apoptotic in the human male germ cell line. Thus, this report is the first to show an anti-apoptotic role of NANOS1 exerted by negative regulation of mRNAs of pro-apoptotic genes.

Mammalian Pumilio (PUM) proteins are sequence-specific, RNA-binding proteins with wide-ranging roles, including germ cell development that has functional implications in fertility. Although human PUM1 and PUM2 are closely related to each other and recognize the same RNA binding motif, there is some evidence for functional diversity, particularly related to their roles in fertility. Here, by RNA sequencing (RNA-Seq) approaches, we identified separate mRNA pools regulated by PUM1 and PUM2 proteins in human male germ cells. Using global mass spectrometry-based profiling, we identified distinct PUM1- and PUM2-bound putative protein cofactors, most of them involved in RNA processing. Combinatorial analysis of RNA-Seq and mass spectrometry findings revealed that PUM1 and PUM2 may form distinct RNA-regulatory networks, with different roles in human reproduction and testicular tumorigenesis. Our findings highlight the functional divergence and versatility of PUM paralogue-based post-transcriptional regulation, offering insight into the mechanisms underlying their diverse biological roles and diseases resulting from their dysfunction.

Background: While Nanos-mediated maintenance of germ cells in Drosophila and mice has been related to regulation of apoptosis, the relevance of these findings to human physiology is uncertain. Previously we have described the p.[(Pro34Thr);(Ser83del)] double NANOS1 mutation as associated with an absence of germ cells in the testes of infertile patients. The aim of this study was to identify the mechanism underlying infertility phenotype of patients bearing the NANOS1 mutation. Methods: Constructs encoding a wild-type or mutated NANOS1 protein were used for transfection of TCam-2 cell line, representing male germ cells in culture. Influence of this mutation on cell proliferation was performed using MTS assay while apoptosis and cell cycle were measured by flow cytometry. RNA-Seq analysis including quantitative RT-PCR was conducted for selecting pro-apoptotic genes, repressed by the wild-type NANOS1. Influence of the p.[(Pro34Thr);(Ser83del)] NANOS1 mutation on that repression was investigated by quantitative RT-PCR. Results: We show here that the p.[(Pro34Thr);(Ser83del)] double NANOS1 mutation causes NANOS1 to functionally switch from being anti-apoptotic to pro-apoptotic in the human male germ cell line TCam-2. This mutation disrupts repression of mRNAs encoding pro-apoptotic GADG45A, GADD45B, GADD45G and RHOB factors, which could contribute to an increase in apoptosis. Conclusions: This report underscores the conservation of Nanos from flies to humans as a repressor of pro-apoptotic mRNAs in germ cells, and provides a basis for understanding NANOS1 functions in human reproductive health.