Cell-free cytoplasmic polyadenylation of oogenic RNA.
The products of cell-free ATP incorporation mediated by cytoplasmic fractions prepared from unfertilized sea urchin eggs, anucleate egg halves, nucleate egg halves, emetine-treated fertilized eggs, and four-cell embryos have been characterized to determine to what extent the polymers synthesized are poly(A) and to assess the size distribution of the primers adenylated. As judged by alkaline lability, ribonuclease resistance, and retention on poly(U)-impregnated filters, greater than 92% of the label recovered after RNA extraction is present in poly(A). LiCl fractionation indicates that little, if any, free poly(A) is synthesized or cleaved from RNA primers during the reaction, and that 4S RNA is not an effective initiator. In excess of 85% of the poly(A) is associated with RNA having S-values greater than or equal to 18S. Sedimentation profiles of RNA adenylated in the unfertilized egg and anucleate egg half reactions are identical. Suppression of in vivo protein synthesis by emetine alters the profile of RNA subsequently adenylated in vitro. It is proposed that the apparent constraints on the utilization of cytoplasmic RNA or ribonucleoprotein primers of oogenic origin may be effected by RNA-associated proteins capable of regulating the selection and/or extent of their polyadenylation during early embryogenesis.