RNA structure determination by solid-state NMR spectroscopy

Alexander Marchanka,Gerhard Althoff-Ospelt,Teresa Carlomagno,Bernd Simon

doi:10.1038/ncomms8024

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.

Highlights

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
Solid-state NMR spectroscopy, which is applicable to macromolecules of any size in non-crystalline form, has emerged as a powerful alternative to study the structure of amyloid fibrils[1,2], membrane proteins[3,4], and large protein– protein assemblies[5]
We demonstrate that RNA structure is accessible at high resolution by solid-state NMR (ssNMR) using a few, easy to prepare, nucleotide-type selectively labeled samples

Summary

G21 U20 G4

Structural determination of the RNA by ssNMR. The determination of RNA secondary structure requires the identification of base pairs. We obtained distance restraints from four different correlation experiments: 13C,15N-TEDOR-13C, 13C-PDSD recorded at multiple mixing times provided carbon– carbon distances; 13C,31P-TEDOR and 13C-band-selective, 15N-TEDOR yielded a few carbon–phosphorus (17) and carbon– nitrogen (6) distances, respectively; CHHC and NHHC experiments yielded distances between protons (Supplementary Table 2). In this context, we proved the applicability of more sophisticated and selective transfer schemes, such as PAR (Proton-Assisted-Recoupling) and PAIN (Proton-AssistedInsensitive-Nuclei)[23,24]. The structures bundles were consistent upon random removal of up to 20% of the total restraints

Discussion

G21 A18 C17

Methods