ja9b10939_si_001.pdf (7.78 MB)
NMR Chemical Exchange Measurements Reveal That N6‑Methyladenosine Slows RNA Annealing
journal contribution
posted on 2019-12-16, 05:31 authored by Honglue Shi, Bei Liu, Felix Nussbaumer, Atul Rangadurai, Christoph Kreutz, Hashim M. Al-HashimiN6-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 R1ρ 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 koff by ∼1-fold but significantly slows the rate of duplex annealing,
decreasing kon 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.