Requirement for RNA-binding activity of MSY2 for cytoplasmic localization and retention in mouse oocytes.
MSY2, a mouse germ cell-specific Y-box protein, is implicated in the global regulation of the stability and/or translation of maternal mRNAs in the mouse oocyte. We report here that in the oocyte approximately 75% of MSY2 protein is associated with a Triton-insoluble preparation, whereas in either male germ cells or when exogenously expressed in transfected somatic cells almost all MSY2 is soluble. This retention in the oocyte, which is unlikely mediated either by microfilaments or by microtubules, markedly decreases beyond the two-cell stage of development. By microinjecting mutant MSY2-EGFP chimeric mRNAs into mouse oocytes and then assaying the expressed protein's localization by laser-scanning confocal microscopy, we find that an intact cold-shock domain (CSD), containing two RNA-binding motifs, is required to localize MSY2 to the oocyte cytoplasm. In addition, an additional basic/aromatic amino acid island (B/A), which can also interact with RNA, in the C-terminal tail domain is necessary to retain MSY2 following Triton permeabilization. Intact mRNA appeared required for this retention, since RNase A treatment of Triton-permeabilized oocytes or microinjection of RNase A into the oocyte released essentially all of the endogenous MSY2 protein. Furthermore, there is a positive correlation between the ability of the mutant MSY2-EGFP protein to remain associated with the Triton-insoluble preparations and its increased affinity for RNA, as determined by RNA electrophoretic mobility shift assays. These results suggest that binding of intact maternal mRNA by MSY2 is required for its cytoplasmic retention.
Yu
Junying
J
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.
Hecht
Norman B
NB
Schultz
Richard M
RM
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002970:Cleavage Stage, Ovum / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005314:Embryonic and Fetal Development; D005260:Female; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013094:Spermatozoa / Q000378:metabolism; D014162:Transfection
D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C116047:Ybx2 protein, mouse