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 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.