N6-Methyladenosine (m6A) residues, which are found internally in viral and cellular mRNA populations at the sequences Apm6ApC and Gpm6ApC, have been proposed to play a role in mRNA processing and transport. We have developed a sensitive approach to analyze the level and location of m6A in specific purified cellular mRNAsin an attempt to correlate m6A location with function. Polyadenylylated mRNA is hybridized to cDNA clones representing the full size mRNA under study or fragments of it, and the protected RNA is digested andlabeled with polynucleotide kinase in vitro. After enrichment for m6A with anti-m6A antibody, the [32P]-pm6A is separated on TLC plates, and compared with the total amount of radiolabeled nucleotides. Using this combination of in vitro RNA labeling and antibody selection, we were able to detect m'A in purified stable mRNAs that cannot be readily labeled in cells with greater sensitivity than was possible by previous techniques. We applied this technique to bovine prolactin mRNA and showed that this mRNA contains m6A. Moreoverj all of the m'A residues in this message are found within the 3' two-thirds of the molecule and are highly concentrated (61%) within a sequence of 108 nucleotides at the 3' noncoding region of the message. The nonrandom distribution of m6A in a specific cellular mRNA, as demonstrated for bovine prolactin, will have to be taken into account when designing a model for m6A function.The most prevalent internal methylated nucleoside in eukaryotic mRNA is N6-methyladenosine (m6A). This modified nucleoside is found in RNAs of higher eukaryotic organisms (1-6), plants (7-9), and viruses (3,(10)(11)(12), and occurs at two specific sequences: . The high degree of sequence specificity, together with the fact that m6A is conserved during processing of the heterogeneous nuclear RNA in the nucleus (11,16,18,19), argues for an important biological function. In viral RNA, internal m6A is distributed in a nonrandom fashion. In Rous sarcoma virus, these methylated nucleosides are concentrated in a 3500-nucleotide region of the 10,000-nucleotide RNA genome (12). The highest level of internal m6A residues in simian virus 40 (SV40) nuclear RNA is found in RNA transcripts from two specific regions of the viral genome (20).The m6A-containing oligonucleotides of late SV40 16S and 19S mRNAs were further localized op the viral genome (15). The specificity of m6A location in particular regions of viral RNA implies that, apart from sequence specificity, other features of the RNA may influence the location of internal methylation and is suggestive of a biological role for m6A.Further indication of the role of m6A was obtained by inhibition studies. Inhibition of internal methylation of mRNA by cycloleucine in B77 avian sarcoma virus results in accumulation of genome-length RNA and decrease in the levels of spliced viral mRNA (21). Cycloleucine treatment of SV40-infected BSC-1 cells results in reduction of the amount of m6A residues as well as inhibition of SV40 mRNA production, but almost no ...

Antibodies specific for N6-methyladenosine (m6A) and for 7-methylguanosine (m7G) were prepared by immunization of rabbits with nucleoside conjugates of bovine serum albumin (i.e, m6A--BSA and m7G-BSA). Specificity of each antibody was assessed by inhibition of the homologous precipitin reaction with various nucleosides. These analyses revealed that the antibodies elicited in response to m6A--BSA were specific for the N6-methyl moiety of adenosine with minimal or no cross-reactivity with BSA, adenosine, and guanosine. Although a major fraction of antibodies elicited in response to m7G--BSA were specific for m7G, considerable cross-reactivity was observed with BSA. These latter antibodies were removed by affinity chromatography utilizing BSA-Sepharose adsorbent. In similar fashion, antibodies specific for m6A and m7G were isolated by immunospecific adsorption to antigen-coupled Sepharose (e.g. m6A--BSA-Sepharose), eluted, and coupled to Sepharose. The ability of these antibody-coupled adsorbents to retain specific methylated [methyl-3H]nucleosides derived from [methyl-3H]tRNA digests was assessed. Both the anti-m7G and anti-m6A antibody adsorbents quantitatively and exclusively retained 7-[3H]methylguanosine and N6-[3H]methyladenosine, respectively. The application of these adsorbents to fractionate oligonucleotides and nucleic acids is discussed.

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