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

ABSTRACT: N⁶-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_1? 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 N⁶-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.

SUBMITTER: Shi H 

PROVIDER: S-EPMC7807175 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature