Enzymatic Characterization of In Vitro Activity of RNA Methyltransferase PCIF1 on DNA

Abstract

PCIF1 and FTO are a pair of human mRNA cap-specific modification enzymes that have opposing activities. PCIF1 adds a methyl group to the N6-position of 2'O-methyladenosine (A_m), generating N6, 2'O-dimethyladenosine (m⁶A_m), when A_m is the cap-proximal nucleotide. FTO removes the N6-methyl group from m⁶A_m. In addition, FTO has a demethylase activity on a broad spectrum of various RNA substrates, as well as on DNA N6-methyldeoxyadenosine (m⁶dA). While the existence of m⁶dA in mammalian DNA remains controversial, we show here that PCIF1 has significant methylation activity on single stranded DNA deoxyadenosine, double stranded RNA/DNA hybrids, and double stranded DNA, though with lower catalytic efficiency than that on its preferred RNA substrate. PCIF1 has activities in the order ssRNA > RNA/DNA hybrid > ssDNA > dsDNA. We discuss the implications of PCIF1 generation, and FTO removal, of DNA adenine methylation.