METHYLATED 5'-TERMINAL CAPS OF ADENOVIRUS TYPE 2 EARLY MESSENGER-RNA - EVIDENCE FOR AT LEAST 6 5'-TERMINI

The 5′-terminal caps of early adenovirus (Ad) 2 mRNA were isolated, fractionated, and compositionally analyzed. Cultured human KB cells were labeled with l-[methyl-3H]methi-onine and 32Pi from 2 to 7 hr postinfection in the presence of cycloheximide. Labeled RNA was extracted from polysomes and fractionated into poly(A)+ and poly(A)- molecules. Viral mRNA was isolated by hybridization to Ad2 DNA, then digested with RNase T2, and cap 1 [m7G(5′)ppp(5′)NmpNp] and cap 2 [m7G(5′)ppp(5')NmpNmpNp] fractions were isolated by DEAE-Sephadex chromatography in 7 M urea. m7G was shown to be located at the 5′ termini of early Ad2 mRNA by periodate oxidation and β elimination. Cap 1 and cap 2 fractions were fractionated by electrophoresis on DEAE-cellulose paper. The cap 1 fraction separated into three major spots (a, b, and d), in order of increasing negative charge), and the cap 2 fraction into four major spots (a, b, c, and d). The methylated constituents of each spot were determined and used to deduce the number and composition of individual caps. The following results were obtained. Cap 1 structures: Spot a is m7GpppUmpN1; b contains m7GpppUmpN2 (N1 and N2 are different unmethylated nucleosides) and m7GpppAmpN; c is m7GpppN*mpN, m7Gpppm6AmpN, and m7GpppGmpN (N*m is m26Am or an unknown base-modified 2′-O-methyl nucleoside). Cap 2 structures: Spot a is m7Gppp(Ump,N*mp)N1; b contains at least two components, combinations of Gm, Um, N*m, and m6Am; c contains at least two components, combinations of Gm, Um, Am, and m6Am; d is at least two components containing combinations of Cm, Am, and m6Am. No significant differences in methylated constituents were detected in early mRNA repared from infected cells in the presence or absence of cycloheximide. Although we detect at least 13 caps, the minimum number of distinct 5′ termini is 6, because (i) N*m could be m26Am (2′-0-methyl N6,N6-dimethyl adenosine), (ii) m6Am and Am could be undermethylated precursors to m26Am, and (iii) cap 1 structures could represent cap 2 structures without methylation of the penultimate nucleoside. Therefore there may be from 6 to 13 distinct Ad2 early mRNAs, which is interesting considering that only four general areas of the genome (each close to the 3′ end of each early gene block) appear to function as promoters [Berk, A. J., and Sharp, P. A. (1977). Cell12, 45-55; Evans, R. M., et al. (1977). Cell12, 733-739]. Analysis of KB cell mRNA resolved about 12 cap 1 and 10 cap 2 structures. All Ad2 mRNA cap spots resolved by electrophoresis corresponded in mobility and composition to major cap spots found in cell mRNA. Thus, the methylated caps of early mRNA are similar to those of the host cell. In contrast, most late Ad2 mRNA molecules have an identical cap sequence [Gelinas, R. E., and Robert, R. J. (1977). Cell11, 533-544]. © 1979.