UNMASKING MESSENGER-RNA 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 v ertebrate oocytes, many of the quiescent stored mRNAs are recruited in to polysomes. In the clam, Spisula solidissima, such masked messages i nclude the abundant mRNAs encoding cyclin A and the small subunit of r ibonucleotide reductase. We have previously shown that mRNA-specific u nmasking of these two messages can be achieved in vitro, in oocyte cel l-free extracts, by the addition of antisense RNAs corresponding to a fairly short (130-140 nucleotides) segment in their cognate 3' untrans lated regions. We postulated that the antisense RNAs prevented the bin ding of a masking repressor protein (Standart et al., 1990). Here we r eport 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 activati on of translation of the masked mRNAs. The role of p82 phosphorylation in maternal mRNA unmasking was assessed in a novel in vitro activatio n system developed from clam oocytes, based upon the natural rise in p H which accompanies fertilization. Concomitant with mRNA unmasking, se veral kinases, including cdc2 and MAP kinases were activated in this s ystem, as was p82 phosphorylation. Inhibitors of serine/threonine kina ses, including 6-DMAP, staurosporine, and H7 inhibited p82 phosphoryla tion, whereas inhibitors of tyrosine kinases, protein kinase C, cAMP-d ependent protein kinase, and p70(s6k) did not prevent this modificatio n. A specific inhibitor of cdc2 kinase, p27(Kip1), prevented p82 phosp horylation and translational activation, strongly suggesting that p82 modification is required for unmasking. (C) 199)6 Academic Press, Inc.