RNA structure determination by solid-state NMR spectroscopy

Abstract

Knowledge of the RNA three-dimensional structure, either in isolation or as part of RNP complexes, is fundamental to understand the mechanism of numerous cellular processes. Because of its flexibility, RNA represents a challenge for crystallization, while the large size of cellular complexes brings solution-state NMR to its limits. Here, we demonstrate an alternative approach on the basis of solid-state NMR spectroscopy. We develop a suite of experiments and RNA labeling schemes and demonstrate for the first time that ssNMR can yield a RNA structure at high-resolution. This methodology allows structural analysis of segmentally labelled RNA stretches in high-molecular weight cellular machines—independent of their ability to crystallize— and opens the way to mechanistic studies of currently difficult-to-access RNA-protein assemblies.

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

“…Further, it is unique in describing RNA dynamics at atomic resolution (Butcher et al 2000;Carlomagno et al 2013;Chowdhury et al 2006;D'Souza et al 2004;Davis et al 2005;Duszczyk et al 2011;Ferner et al 2009;Furtig et al 2003;HouckLoomis et al 2011;Keane et al 2015;Marchanka et al 2015;Sashital et al 2004;Staple and Butcher 2003;Wacker et al 2011). In fact, dynamics ranging from sub nanoseconds to seconds have been found to be essential to understand RNA function Rinnenthal et al 2011).…”

Structural biology of supramolecular assemblies by magic-angle spinning NMR spectroscopy

“…The inclusion of a z-filter is needed to eliminate artifacts due to 13 C- 13 C J couplings in uniformly labeled systems. TEDOR-derived distance restraints have been applied to a range of systems including structure determination of microcrystalline GB1 by Rienstra and co-workers (Nieuwkoop et al, 2009) and L7Ae-bound Box C/D RNA by Carlomagno and co-workers (Marchanka et al, 2015). Pulse sequences, schematics, and model compound spectra for several through-space correlation methods are presented in Fig.…”

“…Recently several labs have demonstrated that MAS NMR can characterize nucleic acids (Cherepanov et al, 2010; Huang et al, 2012) and protein-nucleic acid interactions (Asami et al, 2013; Asami & Reif, 2013; Marchanka et al, 2015; Marchanka et al, 2013) including studies of protein-DNA interactions in intact bacteriophages (Morag et al, 2014; Sergeyev et al, 2011; Yu & Schaefer, 2008). …”

Bacteriophage Tail‐Tube Assembly Studied by Proton‐Detected 4D Solid‐State NMR

“…We note that our approach may also offer advantages for the study of complex materials such as bone [39] or RNAand RNA-protein complexes,which can be challenging for NMR analysis due to the presence of only four different building blocks. [40,41] We also envisage the application of similar experiments to the analysis of chemicalshift anisotropy (CSA) tensors,w here one additional dimension is needed for the CSA recoupling,a nd the CSA dimension requires large spectral widths.…”