NMR Chemical Exchange Measurements Reveal That N⁶-Methyladenosine Slows RNA Annealing

Abstract

N 6-Methyladenosine (m6A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. 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. Little is known about how m6A and other epitranscriptomic modifications might affect the kinetic rates of RNA folding and other conformational transitions that are also important for cellular activity. Here, we used NMR R 1ρ relaxation dispersion and chemical exchange saturation transfer to noninvasively and site-specifically measure nucleic acid hybridization kinetics. The methodology was validated on two DNA duplexes and then applied to examine how a single m6A alters the hybridization kinetics in two RNA duplexes. The results show that m6A minimally impacts the rate constant for duplex dissociation, changing k off by ∼1-fold but significantly slows the rate of duplex annealing, decreasing k on by ∼7-fold. A reduction in the annealing rate was observed robustly for two different sequence contexts at different temperatures, both in the presence and absence of Mg2+. We propose that rotation of the N 6-methyl group from the preferred syn conformation in the unpaired nucleotide to the energetically disfavored anti conformation required for Watson–Crick pairing is responsible for the reduced annealing rate. The results help explain why in mRNA m6A slows down tRNA selection and more generally suggest that m6A may exert cellular functions by reshaping the kinetics of RNA conformational transitions.

2’-O-methylation alters the RNA secondary structural ensemble

“…Global fitting was carried by sharing pES2 and kex for U23-C6 and U38-N3. Due to degeneracy arising from slow exchange (78), R1ρ data measured for TAR in the absence of Mg 2+ and for TAR-C24U25A35 in the absence and presence of Mg 2+ was fit by fixing the population to the values determined using CEST (Table S6).…”

“…iterations (78). In all cases, the observed peak was assumed to be the GS peak, as the system is in slow exchange regime (55).…”

“…We first used UV melting experiments to assess the impact of the modification on the overall thermal stability of TAR, which is dominated by the GS (population ~ 86%) and to a lesser extent ES1 (population ~13%) (see Methods). Given that other post-transcriptional modifications such as m 6 A have been shown to affect RNA thermal stability and structural dynamics in a Mg 2+ -dependent manner (78,88,89), and that the TAR bulge binds Mg 2+ in a unique conformation (90,91), experiments were performed in the absence and presence of Mg 2+ .…”

“…Here, we expected a greater effect because all three Nm modifications should energetically favor ES2 relative to the GS. To overcome a degeneracy arising due to slow exchange kinetics ( Figure S5), we supplemented the R1ρ data with measurements of chemical exchange saturation transfer (CEST) experiments (79,80), which are better suited for studying slower GS-ES exchange process (78).…”

“…CEST experiment. All the 13 C/ 15 N CEST measurements for probing GS-ES2 exchange were performed on site-specific labeled TAR (without Mg 2+ ) and TAR-C24U25A35 RNA (with and without 1 mM Mg 2+ ) as described previously(78)(79)(80). The spin lock power and offset combinations used in CEST measurements are listed inTable S8.…”