| Autor: |
Shi H; Department of Chemistry , Duke University , Durham , North Carolina 27710 , United States., Liu B; Department of Biochemistry , Duke University School of Medicine , Durham , North Carolina 27710 , United States., Nussbaumer F; Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI) , University of Innsbruck , 6020 Innsbruck , Austria., Rangadurai A; Department of Biochemistry , Duke University School of Medicine , Durham , North Carolina 27710 , United States., Kreutz C; Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI) , University of Innsbruck , 6020 Innsbruck , Austria., Al-Hashimi HM; Department of Chemistry , Duke University , Durham , North Carolina 27710 , United States.; Department of Biochemistry , Duke University School of Medicine , Durham , North Carolina 27710 , United States. |
| Abstrakt: |
N 6 -Methyladenosine (m 6 A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. While m 6 A 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 m 6 A 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 m 6 A alters the hybridization kinetics in two RNA duplexes. The results show that m 6 A 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 Mg 2+ . 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 m 6 A slows down tRNA selection and more generally suggest that m 6 A may exert cellular functions by reshaping the kinetics of RNA conformational transitions. |
