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

Abstract

N6-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.

Measuring thermodynamic preferences to form non-native conformations in nucleic acids using ultraviolet melting

“…An excellent correlation ( r = 0.95) was observed between = − and for N 6 -methylamino isomerization in m 6 A measured using NMR RD ( Fig. 6 C ) ( 73 ). The correlation could be fit with an RMSE of 0.1 kcal/mol and ∼0.7 kcal/mol ( Fig.…”

“…When paired with uridine, the methylamino group in m 6 A isomerizes between anti (major) and syn (minor) conformations, resulting in the loss of a hydrogen bond ( Fig. 6 A ) ( 73 ). This conformational change has been proposed to play roles slowing a variety of biochemical processes that involve duplex melting and annealing ( 69 ).…”

“…Using NMR chemical exchange saturation transfer (CEST) experiments ( 74 ), it was recently shown that the minor non-native m 6 A( syn )-U bp transiently forms with a population of ∼1% ( 73 ). It was also shown that N 6 , N 6 -dimethyladenosine (m 6 2 A), a posttranscriptional modification in transfer RNA (tRNA) ( 75 ), structurally mimics this non-native m 6 A( syn )-U bp ( 73 ). This modification destabilizes the major m 6 A( anti )-U conformation both by disrupting the A(N6)-H6–(O4)T hydrogen bond and through steric collisions (curved dashed lines, Fig.…”

“…We tested delta-melt on two sequences shown previously to form stable duplexes in solution, at bp positions for which the non-native m 6 A( syn )-U bp has been previously characterized using NMR and for which the m 6 2 A modification has been shown to recapitulate the NMR chemical shifts of the non-native m 6 A( syn )-U bp ( 73 ) ( SI Appendix , Fig. S4 ).…”

Elucidating the mechanisms underlying protein conformational switching using NMR spectroscopy

“…Modeling data with the Bloch-McConnell equations can provide unambiguous statements on the mechanism of conformational interconversion, as seen in this case study. However, fitting CEST, CPMG RD and R 1ρ RD data to three-state models is fraught with complications and elaborate grid searches must often be undertaken to ensure that the minimization routines do not get trapped in local minima in the parameter surface [ 86 – 93 ]. In this particular report, fitting to a three-state model is facilitated by the fact that there are several residues with three minor dips in their CEST profile, a clear signature of the co-existence of three conformations in equilibrium.…”