Unscheduled m6A Deposition in RNA via m6ATP Incorporation by DNA Polymerases

TL;DR

This study reveals a new way that m6A modifications in RNA can be created through DNA polymerases using m6ATP, which may be linked to cellular stress.

Contribution

The discovery of DNA polymerases incorporating m6ATP into RNA during gap-filling synthesis as a novel mechanism for unscheduled m6A deposition.

Findings

DNA polymerases pol β and pol η efficiently incorporate m6ATP into RNA during gap-filling synthesis.

m6ATP incorporation is comparable to ATP in efficiency and is governed by a base-stacking mechanism.

m6ATP levels in human cells correlate with oxidative stress biomarkers like 8-oxoGTP.

Abstract

N6-methyladenosine (m6A) is the most abundant modification of mRNA and plays a crucial role in mediating cellular functions, and it is associated with cancer and neurodegenerative diseases. Studies have shown that m6A is predominantly deposited on its consensus motif by the m6A writer proteins RNA methyltransferase METLL3/METLL14. However, it was found that nonconventional m6A deposition by other alternative pathways may also exist and can modulate epitranscriptomic regulation in cells. Thus, understanding the molecular mechanisms underlying nonconventional m6A deposition outside the canonical motifs will provide novel insights into the full scope of the functional impact of m6A. In this study, we discovered that m6ATP was efficiently incorporated by the repairing DNA polymerases pol β and pol η through RNA gap-filling synthesis on an RNA-DNA hybrid. Steady-state kinetics results showed that m6ATP was incorporated into RNA by the DNA polymerases with a comparable efficiency to ATP. AlphaFold3-assisted molecular dynamics simulations further elucidated the structural basis for the DNA polymerases to incorporate m6ATP into the RNA substrates by showing that the enzymes employed the unique base-stacking mechanism to govern the distance between the 3′-OH group of the 3′-terminus nucleotide of the primer and the 5′-α-phosphate of m6ATP to perform their catalysis. Furthermore, we detected a significant amount of m6ATP in human cells. We showed that the m6ATP level was associated with that of the oxidative stress biomarker 8-oxoGTP in cells, suggesting that unscheduled m6A deposition on RNA can be mediated by m6ATP incorporation that is associated with cellular oxidative stress. Our study sheds light on the unscheduled m6A deposition as a potential alternative mechanism for altering epitranscriptomic modifications.

Figures