2’-O-methylation alters the RNA secondary structural ensemble

Abstract

2′-O-methyl (Nm) is a highly abundant post-transcriptional RNA modification that plays important biological roles through mechanisms that are not entirely understood. There is evidence that Nm can alter the biological activities of RNAs by biasing the ribose sugar pucker equilibrium toward the C3′endo conformation formed in canonical duplexes. However, little is known about how Nm might more broadly alter the dynamic ensembles of non-canonical RNA motifs. Here, using NMR and the HIV-1 transactivation response (TAR) element as a model system, we show that Nm preferentially stabilizes alternative secondary structures in which the Nm-modified nucleotides are paired, increasing both the abundance and lifetime of a low-populated short-lived excited state by up to 10-fold. The extent of stabilization increased with number of Nm modifications and was also dependent on Mg 2+ . Through phi (Φ) value analysis, the Nm modification also provided rare insights into the structure of the transition state for conformational exchange. Our results suggest that Nm could alter the biological activities of Nm-modified RNAs by modulating their secondary structural ensembles as well as establish the utility of Nm as a tool for the discovery and characterization of RNA excited state conformations.

The Importance of the Epi-Transcriptome in Translation Fidelity

“…The chemical impact of Nm on RNA has been investigated by several studies. It has been reported that Nm biases the sugar pucker equilibrium in favor of the C3′-endo conformation of pyrimidines [ 98 ]. Intra-residue steric repulsion occurs between the Nm, the 3′-phosphate, and the 2-carbonyl groups in the C2′-endo conformation, favoring the C3′ form.…”

Towards SINEUP-based therapeutics: Design of an in vitro synthesized SINEUP RNA

“…18 At the same time, chemical moieties exposed by the particular tertiary structure of RNA can be crucial for the functionality of the molecule, including for its interaction with RNA-binding proteins 19 and its localization and turnover. 18 Some modifications, such as 2 0 O methyl-ATP (Am), 20 N6 methyl-ATP (m6A), 21 and pseudo-UTP (c), 22 alter substantially the RNA secondary structure, which can result in unmasking binding motifs for RNA-binding proteins. 21 Cells add chemical modifications to newly synthesized RNA molecules during and after transcription, complementing the mere sequence of RNA with a multifaceted layer of functionalities that define the intensively studied field of epitranscriptomics.…”

“… 18 At the same time, chemical moieties exposed by the particular tertiary structure of RNA can be crucial for the functionality of the molecule, including for its interaction with RNA-binding proteins 19 and its localization and turnover. 18 Some modifications, such as 2′O methyl-ATP (Am), 20 N6 methyl-ATP (m6A), 21 and pseudo-UTP (ψ), 22 alter substantially the RNA secondary structure, which can result in unmasking binding motifs for RNA-binding proteins. 21 …”