167. MUSASHI FAMILY OF RNA BINDING PROTEINS: CELL CYCLE REGULATORS IN SPERMATOGENESIS
E. A. McLaughlin A C , B. A. Fraser A C , V. Pye A C , M. Bigland A C , N. A. Siddall B C and G. R. Hime B CA Environmental & Life Sciences, University of Newcastle, Callaghan, NSW, Australia.
B Anatomy and Developmental Biology, University of Melbourne, Parkville, VIC, Australia.
C ARC Centre of Excellence in Biotechnology & Development, Australia.
Reproduction, Fertility and Development 22(9) 85-85 https://doi.org/10.1071/SRB10Abs167
Published: 6 September 2010
Abstract
Mammalian meiosis is a tightly regulated process involving specialized cell cycle progression and morphogenetic changes. We have demonstrated that the Musashi family of RNA binding proteins is implicated in the regulation of spermatogonial stem self renewal and germ cell differentiation. Here we describe the novel mechanism by which the Musashi family proteins, Msi1 and Msi2, act to control exit from spermatogonial mitotic amplification and normal entry into meiosis. Gene and protein analysis indicated overlapping Msi1 and Msi2 profiles in enriched populations of isolated germ cells and reciprocal subcellular expression patterns in spermatogonia and pachytene spermatocytes/ round spermatids in testes sections. Recombinant Msi1 protein-RNA pulldown and microarray analysis coupled with in vitro shRNA knockdown studies in spermatogonial culture and subsequent immunoprecipitation and qPCR established that Msi1 targeted Msi2 mRNA for post transcriptional translational repression. Immunoprecipitation of Msi2 target mRNA and subsequent qPCR together with in vitro shRNA knockdown studies in round spermatid culture identified a cell cycle inhibitor protein CDKN1C (p57kip2) as the principal target of Msi2 translational inhibition. Immunolocalisation of CDKN1C protein indicated that expression of this cell cycle regulator coincided with the nuclear import of Msi1 and the appearance of cytoplasmic Msi2 expression in early pachytene spermatocytes. Using a transgenic Msi1 overexpression mouse model in conjunction with quantitative gene and protein expression, we confirmed Msi1 targeting of Msi2 and subsequent Msi2 targeting of CDKN1C for translational repression in vivo. Ectopic overexpression of Msi1 in germ cells induces substantial Msi2 downregulation and aberrant CDKN1C expression, resulting in abnormal spermatogenic differentiation, germ cell apoptosis/arrest and sterility. In conclusion, our results indicate a sophisticated molecular switch encompassing cell cycle protein regulation by Musashi family proteins, is required for normal exit from mitotic division, entry into meiosis and post meiotic germ cell differentiation.
