Selective 2′-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA

Abstract

RNA SHAPE chemistry yields quantitative, single nucleotide resolution structural information based on the reaction of the 2′-hydroxyl group of conformationally flexible nucleotides with electrophilic SHAPE reagents. However, SHAPE technology has been limited by the requirement that sites of RNA modification be detected by primer extension. Primer extension results in loss of information at both the 5′ and 3′ ends of an RNA and requires multiple experimental steps. Here we describe RNase-detected SHAPE (Selective 2′-Hydroxyl Acylation analyzed by Protection from Exoribonuclease) that uses a processive, 3′→5′ exoribonuclease, RNase R, to detect covalent adducts in 5′-end labeled RNA in a one-tube experiment. RNase R degrades RNA but stops quantitatively three and four nucleotides 3′ of a nucleotide containing a covalent adduct at the ribose 2′-hydroxyl or the pairing face of a nucleobase, respectively. We illustrate this technology by characterizing ligand-induced folding for the E. coli thiamine pyrophosphate riboswitch RNA. RNase-detected SHAPE is a facile, two-day approach that can be used to analyze diverse covalent adducts in any RNA molecule, including short RNAs not amenable to analysis by primer extension and RNAs with functionally important structures at their 5′ or 3′ ends.

Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation

“…10b–d). For each species, most TPP-induced changes observed in the aptamer domain were similar to those reported for the thiM riboswitch3941. For example, NAI reactivity increased for positions C53, G54, A56, U63 and A80.…”

“…RNA structure can be studied at the nucleotide level using selective 2′-hydroxyl acylation analysed by primer extension (SHAPE)283334353637383940. We adapted this highly informative technique to interrogate the RNA structure in the context of EC-187.…”

Insights into the Regulatory Landscape of the Lysine Riboswitch

“…Chemical probing was performed essentially as described elsewhere 79,80 . RNA was diluted to 100 nM in 10 µL of a buffer of 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 50 mM KCl, 5 mM MgCl 2 and 0.1 mM EDTA.…”

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8

“…Samples were incubated at 37 • C for 15 min. Folded RNA was divided into two equal portions (72 l each) treated with 8 l of 20 mM 1-methyl-7-nitroisatoic anhydride (1M7) (46) (47). Electropherograms were processed using the open-source SHAPEfinder program ver.…”