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.

Figure 1. Sequence of the Pf Box C/D RNA and magnetization transfer schemes.

(a) Sequence and secondary structure of the Pf Box C/D RNA. Helical regions, light blue; k-turn, green; loop and termini, grey. (b–f) Schematic representation of the magnetization transfer schemes used for resonance assignment and distance measurement, shown on nucleotides stretches highlighted in the sequence. A, green; G, cyan; C, sienna; U, magenta. (b) ¹³C,¹⁵N-TEDOR-¹³C,¹³C-PDSD, A,U^lab-RNA. (c) ¹³C, ³¹P-TEDOR, G,U^lab-RNA. (d) ¹⁵N,¹⁵N-RFDR, G,C^lab-RNA. (e) NHHN (dotted) and NHHC (solid), A,G^lab-RNA. (f) ¹³C, ¹⁵N-TEDOR, (G-¹³C,U-¹⁵N)^lab-RNA.

Figure 2. ssNMR spectra for the sequential assignment and measurement of structural restraints.

(a–b) Ribose region of 2D ¹³C,¹⁵N-TEDOR-¹³C,¹³C-PDSD spectra of (a), G,U^lab-RNA and (b) A,U^lab-RNA (mixing time, 700 ms). Intra- and inter-nucleotide correlations are labeled in green and red, respectively. Selected sequential correlations are shown. Partially overlapped guanosines G10, G14, G16 are labeled as G*; non-site-specifically assigned adenosines in the tetra-loop (A11–A13) are labeled as A*. (c) 2D ¹³C,³¹P-TEDOR spectrum of G,U^lab-RNA. (d) 2D ¹⁵N,¹⁵N-RFDR spectrum showing the G-N1/C-N3 correlations for G:C base pairs.

Figure 3. ssNMR structure of the Pf Box C/D RNA.

(a) Overlay of the 10 lowest energy structures of the Pf Box C/D RNA in complex with L7Ae from ssNMR data. Terminal nucleotides 1 and 25–26 are not shown. Colour code as in Fig. 1a. (b) k-turn of the Pf Box C/D RNA, showing the characteristic geometry. Internal loop, green; NC stem, cyan, C stem, light blue. (c) Comparison of the k-turn geometry of the Pf Box C/D RNA obtained by ssNMR (10 lowest energy structures, gray) with that of the crystallographic structure of the Af Box C/D RNA (PDB code 1RLG), red; Pf Box C/D RNA (PDB code 3NMU), blue; Af Box C/D RNA (PDB code 4BW0), green; Ss Box C/D RNA (PDB code 3PLA), magenta.