M6A Reshapes the Folding and Recognition Landscape of RNAs

Abstract

Ribonucleic acid (RNA) is a versatile and dynamic biomolecule that serves as an indispensable component in the central dogma of molecular biology. The realization that RNA plays a wide variety of roles in gene expression and regulation has been accompanied by the discovery that virtually all types of RNA are chemically modified. These modifications have profound effects on RNA metabolism. N6-Methyladenosine (m6A) is an abundant post-transcriptional RNA modification that influences multiple aspects of gene expression. While m6A is thought to mainly function by recruiting reader proteins to specific RNA sites, the modification can also reshape RNA-protein and RNA−RNA interactions by altering RNA structure mainly by destabilizing base pairing. Here we sought to provide a broad and deep description of how m6A reshapes the folding and recognition landscape of RNA, which provides detailed mechanisms via which m6A exerts its biological functions.First, we show that when neighbored by a 5ʹ bulge, m6A stabilizes m6A–U base pairs and global RNA structure by ~1 kcal/mol. The bulge most likely provides the flexibility needed to allow optimal stacking between the methyl group and 3ʹ neighbor through a conformation that is stabilized by Mg2+. A bias toward this motif can help explain the global impact of methylation on RNA structure in transcriptome-wide studies. While m6A embedded in duplex RNA is poorly recognized by the YTH domain reader protein and m6A antibodies, both readily recognize m6A in this newly identified motif. The results uncover potentially abundant and functional m6A motifs that can modulate the epitranscriptomic structure landscape with important implications for the interpretation of transcriptome-wide data. In addition to altering RNA stability, m6A has also been shown to slow the kinetics of biochemical processes involving RNA-RNA interactions. However, little is known about how m6A affects the kinetic rates of RNA folding and conformational transitions that are important for RNA functions. We developed an NMR relaxation dispersion (RD) method to non-invasively and site-specifically measure nucleic acid hybridization kinetics. Using this method, we discovered that m6A selectively slows annealing rate while has minimal impact on melting rate in different sequence contexts and buffer conditions. To understand the mechanism of the m6A-induced slowdown of hybridization, we used NMR RD to dissect the kinetic pathways of duplex hybridization. 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 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. Taken together, these results uncover the important role of m6A on modulating RNA-RNA and RNA-protein interactions through altering RNA structure and dynamics, highlighting the structural-dynamics-function relationship.