RNA-binding proteins (RBP) are important catalysts of post-transcriptional modifications. The RBP Musashi-2 (MSI2) has previously been implicated in regulating stem cell function and cell division with established roles in proliferation and cell fate determination. During the complex process of mammalian spermatogenesis, post-transcriptional modifications driven via such abundantly expressed RBPs are essential for the development of male gametes. We have previously established that disruption of MSI2 during germ cell maturation is detrimental to sperm development and fertility1. Presently we look to extrapolate the fundamental molecular mechanisms and pathways via which MSI2 functions to exert its control over spermatogenesis.
A series of differential gene (microarray) and protein (iTRAC) expression studies were undertaken to establish the molecular pathways and biological processes affected by up-regulation of MSI2 using a testis specific over-expression mouse model. These studies were verified via quantitative PCR, and immunoblot and immunolocalisation analysis. In addition immunoprecipitation assays were performed in order to identify specific MSI2 interactions within the spermatogenic germ cell population.
Of the differentially expressed (DE) genes, the key molecular networks identified were implicated in cell death, cell morphology, cellular growth and proliferation, and the cell cycle. The most relevant biological processes that characterised the DE proteins related to transcriptional control, the cell cycle and cellular proliferation, translation, apoptosis, RNA splicing/processing, and spermatogenesis. Interestingly there was little to no overlap of individual identities from the two groups. Protein-immunoprecipitation studies identified that MSI2 acts in complex with splicing factor, SFPQ, and piRNA component, PIWIL1, within post-meiotic germ cells.
Overall this data firmly establishes MSI2 as a key regulator of germ cell development with a strong connection to major constituents of the cell cycle. Furthermore, we propose a potential mechanism by which MSI2, via direct interactions with SFPQ and PIWIL1, assists in mRNA processing and translational regulation during spermatogenesis.