RNA binding to human METTL3-METTL14 restricts <i>N</i>⁶-deoxyadenosine methylation of DNA in vitro.

Abstract

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from <i>S</i>-adenosyl-L-methionine to <i>N⁶</i> amino group of adenosine bases in RNA (m⁶A) and DNA (m⁶dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m⁶dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m⁶A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.