MSY2 and MSY4 Bind a Conserved Sequence in the 3 Untranslated Region of Protamine 1 mRNA In Vitro and In Vivo

The mouse Y-box proteins MSY2 and MSY4 are members of a protein family found in prokaryotes and eukaryotes that contain the highly conserved cold-shock domain (CSD). This 67- to 80-amino-acid (aa) nucleic acid binding domain is 43% identical from Escherichia coli to humans, contains the RNP1 and RNP2 RNA binding motifs, and forms a five-stranded antiparallel β-barrel structure (36). The prokaryotic members of this family, such as major cold-shock protein CspA in E. coli, are about 70 aa in length and are involved in the cold-shock response in various bacteria, an adaptive response to sudden temperature downshifts (14, 19). CspA negatively regulates its own expression by acting as an RNA chaperone that destabilizes secondary structures in mRNA (1, 17). The eukaryotic branch of the CSD family are Y-box proteins and have been characterized in all eukaryotes investigated, including fruit flies, planaria, goldfish, chickens, frogs, mice, and humans, with the exception of the yeast Saccharomyces cerevisiae (25, 30). Y-box proteins are approximately 250 to 350 aa in length, have a CSD located in the amino-terminal half of the protein, and possess amino termini which are highly divergent in sequence and length (36). The carboxy tails of invertebrate Y-box proteins are quite variable in structure, with Drosophila melanogaster YPS containing RGG repeats, Caenorhabditis elegans LIN-28 containing zinc fingers, and Schistosoma mansoni SMYB1 containing a fibroin-like domain. On the other hand, the nucleic acid binding carboxy tails of vertebrate Y-box proteins are more conserved and contain four sets of alternating basic and acidic regions, each approximately 30 aa in length (25). Y-box proteins were originally isolated based on their ability to bind the double-stranded (dsDNA) sequence 5′-CCAAT-3′ (29). Later work defined the Y-box element as 5′-CTGATTGG(C/T)(C/T)AA-3′, which contains a reverse CCAAT box (in boldface). This regulatory element is found in the promoter regions of many vertebrate gamete-specific genes, including the Xenopus laevis oocyte-specific hsp70 gene, the rat testis-specific histone H2B gene, and the murine testis-specific Prm1 gene (37). Subsequent work has shown that some Y-box family members have specificity for binding both pyrimidine-rich dsDNA and single-stranded DNA (16). These observations have led to several models for Y-box protein function involving DNA interactions, such as roles in transcriptional regulation, chromatin modification, and DNA repair. Many Y-box proteins have also been identified as components of messenger ribonucleoprotein particles (mRNPs). Y-box protein p50 is the major core protein of cytoplasmic mRNPs of somatic cells in rabbits (10). p50 and the poly(A) binding protein are the two most abundant proteins in these mRNPs. In Xenopus oocytes, Y-box proteins are also abundant components of mRNP3+4s containing masked mRNAs (26). Proteins homologous to mRNP3+4s are expressed during murine spermatogenesis and form complexes with stored mRNAs (21). Murine Y-box protein MSY1 has also been shown to be associated with germ cell mRNPs during spermiogenesis (34). MSY2 and MSY4 are components of a 48- and 50-kDa RNA binding activity present in murine testis extracts (8). This activity is a component of testis mRNPs containing protamine mRNAs. The protamines are small arginine-rich proteins involved in condensation of DNA in the nuclei of mature spermatids. The protamine mRNAs are synthesized in round and early-elongating spermatids, transported to the cytoplasm, and stored as translationally repressed mRNPs until their translation from 2 to 8 days later in elongated spermatids (2, 20). The MSY2 and MSY4 proteins bind a 22-nucleotide (nt) region of the Prm1 3′ untranslated region (UTR) and a 20-nt region of the Prm2 3′ UTR (11). The 22-nt Prm1 region lies within the first 37 nt of the Prm1 3′ UTR and can delay the translation of an hGH transgene in vivo (12). MSY2 is the murine orthologue of Xenopus protein FRGY2 (mRNP3+4) and was cloned from an expression library screen with anti-FRGY2 antibodies (15). FRGY2 was originally cloned by its ability to bind the CCAAT element (35), but as mentioned above is also found associated with germ line mRNPs. FRGY2 is therefore considered to have a dual function: a role in transcriptional activation of oogenic genes and a second role in masking gametogenic mRNAs. Msy4 was cloned from a mouse testis cDNA library using the yeast three-hybrid system with the first 37 nt of the protamine 1 (Prm1) 3′ UTR (Prm11–37wt) as bait (8). The spatial and temporal patterns of both MSY2 and MSY4 are consistent with these proteins playing roles in Prm1 mRNA storage. In this study, we have delineated a site present in the 3′ UTR of the Prm1 3′ UTR that murine Y-box proteins MSY2 and MSY4 bind specifically. Single-nucleotide mutations within the conserved Y-box recognition site (YRS) eliminate binding of MSY2 and MSY4 in vitro and in the yeast three-hybrid system. Furthermore, transgenic experiments with mice suggest that the YRS will also function in vivo.