Two-dimensional chemical mapping for non-coding RNAs


Non-coding RNA molecules fold into precise base pairing patterns to carry out critical roles in genetic regulation and protein synthesis. We show here that coupling systematic mutagenesis with high-throughput SHAPE chemical mapping enables accurate base pair inference of domains from ribosomal RNA, ribozymes, and riboswitches. For a six-RNA benchmark that challenged prior chemical/computational methods, this mutate-and-map strategy gives secondary structures in agreement with crystallographic data (2% error rates), including a blind test on a double-glycine riboswitch. Through modeling of partially ordered RNA states, the method enables the first test of an ʻinterdomain helix-swapʼ hypothesis for ligand-binding cooperativity in a glycine riboswitch. Finally, the mutate-and-map data report on tertiary contacts within non-coding RNAs; coupled with the Rosetta/FARFAR algorithm, these data give nucleotide-resolution three-dimensional models (5.7 Å helix RMSD) of an adenine riboswitch. These results highlight the promise of a two-dimensional chemical strategy for inferring the secondary and tertiary structures that underlie non-coding RNA behavior. 3 The transcriptomes of living cells and viruses continue to reveal novel classes of non-coding RNA (ncRNA) with critical functions in gene regulation, metabolism, and pathogenesis (see, e.g., refs 1-7). The functional behaviors of these molecules are intimately tied to specific base-pairing patterns that are challenging to identify by existing strategies based on phylogenetic analysis, NMR 8-11 , crystallography 12-17 , molecular rulers 18, 19 , or functional mutation/rescue experiments (see, e.g., refs 7, 20, 21). A more facile approach to characterizing RNA structure involves high-throughput chemical mapping at single-nucleotide resolution. This method is applicable to RNAs as large as the ribosome or entire viruses both in vitro and in their cellular milieu. 22-25 Measurements of every nucleotideʼs accessibility to solution chemical modification can guide or filter structural hypotheses from computational models 26-28. Nevertheless, approximations in computational models and in correlating structure to chemical accessibility limit the inherent accuracy of this approach. 26-31 This paper presents a strategy to expand the information content of chemical mapping through a two-dimensional ʻmutate-and-mapʼ methodology. 32 Here, sequence mutation acts as a second dimension in a manner analogous to initial perturbation steps in multidimensional NMR pulse sequences 11 or pump/probe experiments in other spectroscopic fields 33. We reasoned that if one nucleotide involved in a base pair is mutated, its partner might become more exposed and thus be readily detectable by chemical mapping. In practice, some mutations might not lead to the desired ʻreleaseʼ of the pairing partners; and some mutations …

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@inproceedings{Kladwang2011TwodimensionalCM, title={Two-dimensional chemical mapping for non-coding RNAs}, author={Wipapat Kladwang and Christopher C. VanLang and Pablo Cordero and Rhiju Das}, year={2011} }