FIG. 8.
Polyadenylation and N-7 methylation enhance translational recruitment of LUC/B1wt mRNA in the absence of cap ribose methylation. (A) Stimulation of luciferase activity during oocyte maturation. Oocytes were collected within 1 h of maturation, extracts were prepared, and luciferase activity was determined. The data represent the averages of two separate samples (five oocytes each) for each point. Values of luciferase activity (expressed as arbitrary units per amount of mRNA injected) are shown. Samples were collected immediately following oocyte injection (0) or after maturation (approximately 3 h) following incubation in the absence (−P) or presence (+P) of progesterone. The ratio of luciferase activity observed in mature versus nonmature oocytes (+P/−P) is indicated. (B) The fraction of LUC/B1wt mRNA associated with polysomes increases following maturation. Cap-labeled LUC/B1wt and LUC/B1mut mRNAs were synthesized and manipulated as described in the text. Following incubation in the absence or presence of progesterone, oocytes were homogenized, and samples were separated across a 10 to 50% linear sucrose gradient. The percentage of total counts per minute present in each fraction (y axis) is plotted against the fraction number. Only fractions 1 through 10 (of 12 fractions total) are shown. A representative A254 tracing is depicted as a dashed line. ▴, LUC/B1wt mRNA, and ◊, LUC/B1mut, mRNA incubated in the absence of progesterone; ○, LUC/B1wt mRNA, and ⧫, LUC/B1mut, mRNA injected, progesterone added. (C) Polyadenylated LUC/B1wt mRNA becomes polysome associated in the absence of cap ribose methylation. Fractions from the experiment were pooled to reflect polysome-associated (larger than the 80S monosome peak; fractions 1 through 6) and nonpolysome-associated regions of the gradient (fractions 7 through 12). LUC/B1wt mRNA was extracted, digested with nuclease P1, and analyzed by 2D TLC. A portion of the combined fractions 1 through 6 was further selected by binding to oligo(dT) cellulose prior to P1 digestion. Part 1, not injected into oocytes; part 2, progesterone added, nonpolysomal fractions 7 through 12; part 3, progesterone added, polysomal fractions 1 through 6; part 4, progesterone added, oligo(dT)-selected mRNA from polysomal fractions 1 through 6. Nucleoside 5′-monophosphates and cap dinucleotides are as indicated in the legend for Fig. 1. The position of the ribose methylated cap, m7G*pppGm, is indicated by the arrowhead.