Functional analysis of the murine sequence-specific RNA binding protein MSY4

The translational regulation of the protamine genes has been extensively studied as a model for translational control. The protamines are small arginine-rich proteins involved in DNA condensation in spermatids. The protamine mRNAs are maximally synthesized in round spermatids, stored in the cytoplasm as translationally repressed messenger ribonucleoprotein particles (mRNPs) for up to 10 days, and then translated in elongated spermatids. Premature translation of murine protamine 1 (Prm1) causes premature nuclear condensation, inhibition of spermatid differentiation, and dominant male sterility. Studies with transgenic mice have shown that the 3′ untranslated region (UTR) of Prm1 mRNA is both necessary and sufficient for proper translational control. Though many particulars of protamine translational control have been ascertained, the mechanism of this repression has not been fully elucidated.; This dissertation focuses on studies involving MSY4, a Y-box protein that specifically binds a cis element in the Prm1 and Prm2 3′ UTRs. I have characterized the components of a MSY4-containing testis-specific RNA binding activity. MSY4 is expressed in the cytoplasm of pachytene spermatocytes and round spermatids; the correct spatial and temporal expression pattern for a repressor of protamine translation. Polysome analysis demonstrated that MSY4 is associated with mRNPs, consistent with MSY4 having a role in storing repressed messages. I have extensively characterized the MSY4 binding site, both in vivo and in vitro, by electrophoretic mobility shift assays and the yeast three-hybrid system. This work has defined a 7-nucleotide consensus sequence that is required for MSY4 RNA-binding. Mutation of this site relieves translational repression of a transgenic reporter mRNA in vivo. Finally, gain-of-function studies of MSY4 have been carried out employing transgenic mice that express MSY4 in the germ line. Two transgenic lines were generated that extend MSY4 expression beyond that of the endogenous gene. Both lines of transgenic mice exhibit dominant male sterility and abnormal sperm development. Analysis of candidate gene expression by immunohistochemistry in these mice indicated that MSY4 can behave as a repressor of translation in a message-specific manner.