Talk abstracts
Talk on Friday 01:15-01:30pm submitted by David Mitchell III
In vivo RNA structural probing of uracil and guanine base pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
David Mitchell III (Department of Chemistry, Penn State University), Andrew J. Renda (Department of Biochemistry and Molecular Biology, Penn State University), Catherine A. Douds (Department of Chemistry, Penn State University), Paul Babitzke (Department of Biochemistry and Molecular Biology, Penn State University), Sarah M. Assmann (Department of Biology, Penn State University), Philip C. Bevilacqua (Department of Chemistry, Penn State University)
Abstract:
Many biological functions performed by RNAs arise from their in vivo structures. Chemical reagents that modify the Watson-Crick (WC) face of unprotected RNA bases report on the absence of base pairing and so are of value to determining structures adopted by RNAs. The structure of the same RNA can differ in vitro and in vivo owing in part to the influence of molecules, ranging from protons to secondary metabolites to proteins. Reagents have thus been sought that can report on the native RNA structures that prevail in living cells. Dimethyl sulfate (DMS) and glyoxal penetrate cell membranes and inform on RNA secondary structure in vivo through modification of adenine (A), cytosine (C), and guanine (G) bases. Uracil (U) bases, however, have thus far eluded characterization in vivo. Herein, we show that the water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is capable of modifying the WC face of U and G in vivo, favoring the former nucleobase by a factor ~1.5, and doing so in the eukaryote rice, as well as in the Gram-negative bacterium Escherichia coli. While both EDC and glyoxal target Gs, EDC reacts with Gs in their typical neutral state, while glyoxal requires Gs to populate the rare anionic state. EDC may thus be more generally useful; however, comparison of the reactivity of EDC and glyoxal may allow the identification of Gs with perturbed pKas in vivo and genome-wide. Overall, use of EDC with DMS allows in vivo probing of the base pairing status of all four RNA bases.
References:
Bevilacqua PC, Assmann SM. 2018. Technique development for probing RNA structure in vivo and genome-wide. In Additional Perspectives on RNA Worlds, (ed. TR Cech, JA Steitz, JF Atkins). Cold Spring Harbor Laboratory Press, New York, NY (in press).
Mitchell D, 3rd, Ritchey LE, Park H, Babitzke P, Assmann SM, Bevilacqua PC. 2018. Glyoxals as in vivo RNA structural probes of guanine base-pairing. RNA 24: 114-124.
Ritchey LE, Su Z, Tang Y, Tack DC, Assmann SM, Bevilacqua PC. 2017. Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo. Nucleic Acids Res.
Rouskin S, Zubradt M, Washietl S, Kellis M, Weissman JS. 2014. Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505: 701-705.
Keywords: in vivo RNA probing, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, RNA structure