Mapping of N6-methyladenosine residues in bovine prolactin mRNA.

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Abstract
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 mRNAs in 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 and labeled 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 m6A 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. Moreover, all of the m6A residues in this message are found within the 39 two-thirds of the molecule and are highly concentrated (61%) within a sequence of 108 nucleotides at the 39 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.