Unmasking mRNA in clam oocytes: role of phosphorylation of a 3' UTR masking element-binding protein at fertilization.
During meiotic maturation or after fertilization of invertebrate and vertebrate oocytes, many of the quiescent stored mRNAs are recruited into polysomes. In the clam, Spisula solidissima, such masked messages include the abundant mRNAs encoding cyclin A and the small subunit of ribonucleotide reductase. We have previously shown that mRNA-specific unmasking of these two messages can be achieved in vitro, in oocyte cell-free extracts, by the addition of antisense RNAs corresponding to a fairly short (130-140 nucleotides) segment in their cognate 3' untranslated regions. We postulated that the antisense RNAs prevented the binding of a masking repressor protein (Standart et al., 1990). Here we report UV-crosslinking and gel retardation studies which show that the masking portions of the translationally regulated mRNAs bind an oocyte protein of 82 kDa (p82), which is phosphorylated after fertilization. This modification was accompanied by altered RNP complex formation in gel retardation assays. These changes presumably reflect the activation of translation of the masked mRNAs. The role of p82 phosphorylation in maternal mRNA unmasking was assessed in a novel in vitro activation system developed from clam oocytes, based upon the natural rise in pH which accompanies fertilization. Concomitant with mRNA unmasking, several kinases, including cdc2 and MAP kinases were activated in this system, as was p82 phosphorylation. Inhibitors of serine/threonine kinases, including 6-DMAP, staurosporine, and H7 inhibited p82 phosphorylation, whereas inhibitors of tyrosine kinases, protein kinase C, cAMP-dependent protein kinase, and p70s6k did not prevent this modification. A specific inhibitor of cdc2 kinase, p27Kip1, prevented p82 phosphorylation and translational activation, strongly suggesting that p82 modification is required for unmasking.
Walker
J
J
Department of Biochemistry, University of Cambridge, Massachusetts.
Dale
M
M
Standart
N
N
D000595:Amino Acid Sequence; D000818:Animals; D049872:Bivalvia / Q000201:enzymology / Q000235:genetics*; D016203:CDC2 Protein Kinase / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002474:Cell-Free System; D016213:Cyclins / Q000235:genetics; D004789:Enzyme Activation; D005306:Fertilization / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000235:genetics
D016213:Cyclins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011189:Potassium Chloride; D012264:Ribonucleotide Reductases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016203:CDC2 Protein Kinase