N6-methyladenosine (m6A) residues are present as internal base modifications in most higher eucaryotic mRNAs; however, the biological function of this modification is not known. We describe a method for localizing and quantitating m6A within a large RNA In addition to having 5'-methylated cap structures, many eucaryotic mRNAs contain internal methylated bases. N6-methyladenosine (m6A), the major form of modified nucleoside, has been detected in most higher eucaryotic cell mRNAs, in viral mRNAs, and in the virion RNA of retroviruses (2). Methylation of internal adenosines occurs in the nucleus very rapidly after synthesis of the heterogeneous nuclear RNA precursor to mRNA and before the RNA is spliced (9, 28). While the function of this internal methylation is not known, several observations suggest a significant role for m6A in RNA metabolism.Some implications for the function of internal base modifications come from methylation inhibition studies, using either cycloleucine or S-tubercidinylhomocysteine to block internal methylation of RNA. These experiments suggest an involvement of m6A in RNA processing events or in the transport of mRNA from the nucleus to the cytoplasm (6, 13, 35). It is not clear from these studies, however, that the effect of the inhibitors is directly related to the methylation state of the mRNA being examined. Secondary effects of the drugs have not been ruled out. Furthermore, the requirement for internal methylation in mRNA is not absolute, as mature messages without any detectable m6A residues do exist, including globin and histone mRNAs (24,27).In all cases characterized to date, m6A occurs only in the sequences Gm6AC and Am6AC (7,12,31,38). Preliminary localization studies with specific RNAs have determined that the distribution of m6A within a molecule is nonrandom.Rous sarcoma virus (RSV), for example, has an average of 10 to 15 m6A residues per virion RNA molecule (4, 14, 36).These methylated bases have been detected only in the 3' half of the genomic RNA (4), even though sequence analysis shows that putative methylation sites (GAC and AAC) are widely dispersed throughout the molecule (32). Likewise, internal methylation of bovine prolactin mRNA is confined to the 3' two-thirds of the message, with a high concentration of m6A in the 3' noncoding region (17). Simian virus 40 * Corresponding author.(SV40) late mRNAs, on the other hand, each contain an average of three m6As, all near the 5' ends of those messages in translated sequences (7). Adenovirus mRNAs are methylated in the 5' two-thirds of the molecules, and m6As seem, in this system, to be conserved during mRNA processing (9, 33).We have developed a method for directly identifying m6A residues within the genomic RNA of RSV. We have precisely mapped seven such sites in the genome, all within protein coding regions. In addition, we have analyzed the extent of modification at individual methylation sites. Earlier studies with SV40 (7) and with RSV (12) suggest that methylation at some specific sites is heterogeneous, with bas...