Analysis andin vitrolocalization of internal methylated adenine residues in dihydrofolate reductase mRNA

A T7 RNA transcript coding for mouse dihydrofolate reductase (DHFR) was utilized as a substrate for the N6-methyladenosine mRNA methyltransferase isolated from HeLa cell nuclei. This transcript acted as a 3 fold better substrate than either prolactin mRNA or a synthetic RNA substrate which contained multiple methylation consensus sequences. Formation of internal N6-methyladenine (m6A) residues in the DHFR transcript was shown to increase slightly by the absence of a 7-methylguanine-2'-O-methyl cap structure. Using T7 transcripts from different regions of the DHFR gene, the majority of the m6A residues were localized to the coding region and a segment of the transcript just 3' to the coding region. This data suggests that DHFR mRNA contains multiple methylation sites with most of these sites concentrated in the coding region of the transcript.