N-6-METHYL-ADENOSINE IN ADENOVIRUS TYPE-2 NUCLEAR-RNA IS CONSERVED IN THE FORMATION OF MESSENGER-RNA

In the biogenesis of adenovirus type 2 messenger RNAs, methylation occurs at the 5′ end (cap) and to internal adenosine residues to yield N6-methyl-adenosine (m6A) (Sommer et al., 1976; Moss & Koczot, 1976; Wold et al., 1976). The kinetics of accumulation of 3H from methyl-labeled methionine and 14C from uridine into Ad-2† † Abbreviation used: Ad-2, adenovirus type 2.-specific RNA was measured late in Ad-2 infection. As reported previously (Nevins & Darnell, 1978a), the rate of accumulation of [14C]uridine label in nuclear RNA was approximately four- to fivefold faster than in the cytoplasmic RNA, indicating a conservation of about 20% for the total RNA. The initial rates of [3H]methyl label in m6A in nuclear RNA and in the cytoplasmic RNA were approximately equal, suggesting a complete (or nearly complete) conservation of m6A. In accord with the accumulation kinetics, the ratio of 3H to 14C was higher in cytoplasmic RNA than in nuclear RNA that hybridized to equivalent regions of the Ad-2 DNA. A mathematical model was designed to evaluate the accumulation of methyl label in m6A, taking into consideration the three major parameters that affect the accumulation curves: equilibration of the S-adenosyl-methionine pool, the nuclear dwell time of sequences destined to be mRNA, and the cytoplasmic stability of mRNA. The half-time (t 1 2) for pool equilibration was determined experimentally to be 22 minutes and the nuclear dwell time and the mean life-time of cytoplasmic mRNA were estimated from 14C label to be about 30 and 70 minutes, respectively. The model gave an excellent fit to the data when the t 1 2 for pool equilibration time of 24 ± 2 minutes, a nuclear dwell time of 25 ± 10 minutes, and a mean cytoplasmic mRNA life-time of 75 ± 30 minutes were used to evaluate accumulation curves. Even when data from a restricted region of the genome, 40.5-52.6, which encodes the main portion of at least five 3′ co-terminal mRNAs whose spliced junction with the tripartite leader sequence varies from 38, 40, 43, 45, and 48 was analyzed, it appeared that m6A was conserved. Finally, m6A was found to be added in a brief label (3.5 min) mainly to nuclear molecules that were longer than any cytoplasmic RNA. The conservation of m6A and its addition prior to splicing raise the possibility that internal methylations are involved, in the formation of mRNA. © 1979.