Developmental Changes in the Methylation of Silkmoth Follicular Epithelial Messenger Ribonucleic Acid

The methylation of silkmoth follicular epithelial messenger ribonucleic acid (mRNA) was investigated at different stages of follicular development. Follicles from the late prechorionating and four chorionating stages were incubated in organ culture for 2 h with [methyl-3H]methionine. The resulting polysomal-derived, poly(A+) mRNAs were characterized with respect to both size and composition of methyl-3H-methylated constituents. As evaluated by sucrose density gradient centrifugation, prechorion mRNAs were heterogeneous in size (7-30S) in contrast to the relatively homogeneous chorion mRNAs which sedimented within the 7-14S region of the gradient. Analysis of the methyl-3H-labeled products of mRNA digests (nuclease-P, or RNase T2) via DEAE-cellulose and thin-layer chromatographic techniques revealed the following information. First, on the basis of nuclease-P1 digestion, the predominating 5'-terminal cap structure in both prechorion (greater than 70%) and chorion (greater than 90%) mRNAs was m7G(5,)ppp(5')Am, with minimal amounts of m7GpppGm and m7GpppCm (prechorion) and m7Gpppm6Am (chorion) also detected. Second, on the basis of RNase-T2 digestion, the ratio of cap type 1 structures (m7GpppNmpNp) to cap type 2 structures (m7GpppNmpNmpNp) was 1:1 and 7:1 for prechorion and chorion mRNAs, respectively. Additionally, whereas prechorion mRNAs possessed an average of two internal m6A residues per cap, chorion mRNAs were deficient in this or other internal methylated nucleosides, i.e., less than 0.1 m6A/mRNA. As measured by the extent of incorporation of [3H] methyl groups into 5'-terminal m7GpppNm structures, the synthesis of poly(A+) mRNA progressively increased during follicular development, ultimately exceeding the prechorion value by 20-fold. Collectively, these results indicate that significant alterations in the methylation patterns of silkmoth mRNA accompany transitions in the types and amounts of mRNA expressed during follicular development.