RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro.

Shan Qi,Javier Mota,Shailee Arya,Johanna Villarreal,Yogesh K Gupta,Manjeet K Rao,Ivan R Corrêa Jr,Siu-Hong Chan,Robert A Hromas,Nan Dai

doi:10.7554/elife.67150

Abstract

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 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.

Highlights

N6-methyladenosine (m6A) is considered a major covalent modification of the adenosine (A)base in coding and noncoding RNAs1-5
It is linked to diverse physiologic processes, including – but not limited to – RNA turnover[6,7,8], stem cell differentiation[9], translation[11,12,13,14], and DNA damage repair[15], installed by Methyltransferase like-3 (METTL3) and METTL14 methyltransferases[2, 4, 17], both of which belong to the βclass of S-adenosyl methionine (SAM)-dependent methyltransferases (N6-MTases) 18, 19 that include N6-deoxyadenosine DNA methyltransferases (m6dA MTases), especially those from
DNA are held in closed proximity, as supported by recent studies suggesting: a) localization of METTL3 to sites of double-stranded DNA breaks, and its role in DNA/RNA hybrid accumulation[16], b) recruitment of METTL3 to chromatin by promoter-bound transcription factors[12], c) UV-induced DNA damage signaling[30], and d) metabolism of R-loops[45]

Summary

Introduction

N6-methyladenosine (m6A) is considered a major covalent modification of the adenosine (A)base in coding and noncoding (nc) RNAs1-5. While m6dA in bacterial DNA is deposited in a strictly sequence-dependent manner in doublestranded DNA (dsDNA) 20, 23, m6A in RNA by human METTL3-METTL14 shows less stringent sequence dependency for sequences flanking the target A within the recognition motif DRACH (D=A/G/U, R=A/G, H=A/C/U)[2], repertoire of target transcripts of METTL3-METTL14. This does not correlate with low levels of m6A in cellular RNA, which remains at ~ 0.1% of total pool of ribonucleotides in cultured mammalian cells[25]. Recent evidence suggests that all m6A is deposited cotranscriptionally, with most occurring on chromatin-associated RNAs, e.g., nascent pre-mRNA

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Conclusion