A quantitative model predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions

Bei Liu,Kevin Andreas Erharter,Christoph Kreutz,Honglue Shi,Chia-Chieh Chu,Hashim M Al-Hashimi,Felix Nussbaumer,Atul Rangadurai,David A Case

doi:10.1038/s41467-021-25253-8

Abstract

ABSTRACTN6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. The modification also slows biochemical processes through mechanisms that are not understood. Using temperature-dependent (20°C–65°C) NMR relaxation dispersion, we show that m6A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting at elevated temperatures via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes.

Highlights

N6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites
The ssRNAanti which is the intermediate along the conformational selection (CS) pathway has been extensively characterized in the past, whereas there is no evidence for the dsRNAsyn IF intermediate
We developed a CS model which assumes that the minor anti isomer of m6A hybridizes with apparent annealing and melting rate constants similar to those of the unmethylated RNA

Summary

Introduction

N6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. We developed and validated a nuclear magnetic resonance (NMR) relaxation–dispersion (RD)18–20-based method to measure the hybridization kinetics in DNA and RNA duplexes[21] Using this approach, we showed that m6A preferentially slows the apparent rate of RNA duplex annealing by ~5–10-fold while having little effect on the apparent rate of duplex melting[21] (Fig. 1b). The comparable m6A-induced slowdown observed for duplex annealing and a variety of biochemical processes indicates that a common mechanism might be at play[13,16,17] It has been known for many decades that the methylamino group of the m6A nucleobase can form two rotational isomers that interconvert on the millisecond timescale[25,26] (Fig. 1a). As isomerization is energetically disfavored, it has been proposed to explain how m6A destabilizes dsRNA via the so-called “spring-loading”[12] mechanism despite forming a canonical Watson–Crick m6A–U bp

Methods

Results

Conclusion