RNA-binding proteins (RBP) are important facilitators of post-transcriptional gene expression. 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 nuclear overexpression of MSI2 during germ cell maturation is detrimental to sperm cell development and fertility. Herein we look to explore the molecular mechanisms and pathways by which MSI2 functions to exert its control of spermatogenesis.

A series of differential gene and protein expression studies were undertaken to establish the molecular pathways and biological processes affected by up-regulation of MSI2 using a novel testis-specific overexpression mouse model. Of the differentially expressed genes identified through comparative microarray analysis, the key molecular networks identified were implicated in cell death, cell morphology, cellular growth and proliferation, and the cell cycle. While the most relevant biological processes that characterised our comparative protein studies were linked to transcriptional control, the cell cycle and cellular proliferation, translation, apoptosis, RNA splicing/processing, and spermatogenesis. Gene target analysis revealed the MSI2-mediated translational suppression of TBX1and PIWIL1, both confirmed as direct RNA-binding targets of MSI2 during spermatogenesis. Protein-protein interaction studies identified that MSI2 acts in complex with splicing factor SFPQ in testis germ cells, suggesting a potential function for MSI2 in splicing regulation. Overall this data firmly establishes MSI2 as a key regulator of male gamete development via post-transcriptional regulation. Furthermore we propose a mechanism for MSI2 in pre-mRNA processing as a means of translational regulation during spermatogenesis.