Translational control is an important step of gene regulation, especially during gametogenesis and embryogenesis. In meiotic and postmeiotic spermatogenic cells, essentially more than 70% of all mRNAs are present as translationally inactive messenger ribonucleoprotein particles (mRNPs). Y-box RNA-binding proteins are major constituents of mRNPs and have an ability to repress translation of mRNAs non-specifically. Mouse testis contains at least three Y-box RNA-binding proteins, including germ cell-specific MSY2 and MSY4. Male mice lacking MSY2 or MSY4 show reproductive defects at different stages. As a first step to clarify the functional difference(s) between MSY2 and MSY4 at the molecular level, we have characterized the two proteins with a focus on their function in translation.
  Immunoblot and immunocytological analyses of spermatogenic cells revealed that MSY2 persisted until elongating spermatids, whereas MSY4 decreased dramatically at later stages of round spermatids. MSY2 and MSY4 were associated with not only translationally inactive mRNPs but also active polysomes both in testis and in transfected HEK293T cells, as analyzed by sucrose density gradient ultracentrifugation. Luciferase reporter assay and polysome analysis showed that the two proteins are capable of repressing translation in cultured cells. Using deletion mutants of MSY4, it was found that C-terminal domain, which is responsible for non-specific interactions with mRNAs and with poly(A)-binding protein, causes translational repression. However, MSY4 failed to inhibit translation of histone mRNA, implying that poly(A) tail is possibly required for general translational repression. In addition, artificially tethered cold-shock domain of MSY4 also reduced the luciferase reporter activity, suggesting its involvement in regulation of haploid-specific mRNAs carrying Y-box recognition sequence (YRS), including protamine mRNAs. Taken together, our results suggest that MSY2 and MSY4 possess two functional domains; one is C-terminal domain responsible for general translational repression of poly(A)-containing mRNAs and the other is cold-shock domain involved in sequence-specific regulation of YRS-containing mRNAs.