FIG. 2.
Cap ribose methylation of synthetic RNA substrates is independent of polyadenylation. (A) Polyadenylation of 3′UTR RNA substrates during oocyte maturation. Samples of five oocytes each were collected and analyzed. Each lane contains RNA equivalent to one-half oocyte. Cyclin B1wt RNA: lane 1, not injected; lane 2, injected, no progesterone; lane 3, injected, progesterone added; lane 4, injected, progesterone added, selection by oligo(dT) cellulose chromatography. Cyclin B1mut RNA: lane 5, not injected; lane 6, injected, no progesterone; lane 7, injected, progesterone added; lane 8, injected, progesterone added, selection by failure to bind oligo(dT) cellulose. (B) Oocyte maturation-dependent cap methylation of RNAs. Samples from panel A were digested with nuclease P1, and the products were separated by 2D TLC. Each chromatogram contains RNA equivalent to one-half oocyte. Chromatograms 1 through 4 contain RNA present in panel A, lanes 1, 4, 5, and 8, respectively. Cyclin B1(wt) RNA: part 1, not injected; part 2, injected, plus progesterone. Cyclin B1(mut) RNA: part 3, not injected; part 4, injected, plus progesterone. Part 5, legend. Nucleoside 5′-monophosphates and cap dinucleotides are labeled as described in the legend to Fig. 1.