Applications of NMR to structure determination of RNAs large and small

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to investigate the structure and dynamics of RNA, because many biologically important RNAs have conformationally flexible structures, which makes them difficult to crystallize. Functional, independently folded RNA domains, range in size between simple stem-loops of as few as 10-20 nucleotides, to 50-70 nucleotides, the size of tRNA and many small ribozymes, to a few hundred nucleotides, the size of more complex RNA enzymes and of the functional domains of non-coding transcripts. In this review, we discuss new methods for sample preparation, assignment strategies and structure determination for independently folded RNA domains of up to 100 kDa in molecular weight.

Keywords: Isotope labeling; NMR spectroscopy; RNA; Structure.

Fig. 1. PDB statistics of deposited RNA structures as of Dec 2016

(A) Total number of PDB depositions per year and (B) total RNA structures deposited per year. Here, all reported coordinates are shown in blue, X-ray structures are in red, while NMR structures are in green. Structures determined with other techniques (e.g. electron microscopy; EM) are shown with dark brown filled circles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Methods for small RNA preparation

(a) Preparation of RNA based on the incorporation of an HH ribozyme at the 5′-end of the target RNA. The first 10–12 residues of the target RNA are complementary to a guide sequence in the HH ribozyme. The scissors indicates the cleavage site [18]. (b) Alternative methods; double RNase H cleavage and RNase H/VS ribozyme cleavage protocols [23] Both methods use a chimeric DNA sequence to anneal to the RNA template before RNase H cleavage. (c) Diagram of a DNA Enzyme of the “10–23” family shown with single letters core and two flanking regions of 5–12 nts on either side, designed to base-pair to the RNA substrate. The arrow indicates the cleavage site between unpaired purine (Pu) and paired pyrimidine (Py) in the RNA substrate [25].

Fig. 3. NMR Assignment strategy for an RNA thermometer of ~70 nucleotides

(A) Three smaller constructs were prepared; the first corresponds to the upper stem, the second overlaps with the lower stem while the third is representative of the middle region of the RNA. (B) An overlay of the imino regions of 2D ¹H–¹H NOESY spectra. The black color represents the spectrum for the complete RNA, while green, blue and red are from the fragments, upper, lower and middle stem, respectively. The spectra of the fragments match the full RNA almost perfectly. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Effect of site-specific ribose deuteration on the quality of the NMR spectra of an RNA thermometer

(A) secondary structure of the ‘core’ thermometer. (B) and (C) are 2D ¹H–¹H NOESY spectra recorded in D₂O for completely protonated (B) or with H6/H8, H1′, H2′, D3′, D4′, D5′/D5′ and D5 ribose deuteration, respectively, at 25 °C and 600 MHz. Spectral simplification is remarkable.

Fig. 5. Segmental labeling of RNA

(A–B) Overview of the preparation of a segmentally isotope labeled RNA. Here (A) shows the construction of a plasmid where 3′-HH and 5′-HH are engineered. Panel (B) shows the simplification of aromatic regions of ¹H, ¹³C-TROSY spectra for the RNA, the top panel shows spectra for uniformly ¹³C/¹⁵N-labeled RNA (black), middle for 5′¹³C- and 3′¹⁵N-labeled RNA (red) and the bottom spectrum corresponds to 5′¹⁵N- and 3′¹³C- labeled RNA (blue). The figure is taken from Tzakos et al. [54]. (C) Segmental labeling approach based on T4 DNA ligase, DNA splint and T4 RNA ligase adapted from Nelissen et al. [55]. The segmentally labeled RNA can be prepared either in single or multi-step ligations to obtain the desired product. Here, dark black, grey and vertically patterned fill colors represent three RNA fragments to be joined either in a two-step ligation based on T4 DNA ligase/DNA splint/T4 RNA ligase or single step ligation by T4 RNA ligase. Each fragment can be differently labeled or unlabeled. (D) A newly developed segmental labeling approach from Duss et al. [24] based on HH/VS ribozymes, 2′-O-methyl RNA/DNA chimera, RNase H and T4 DNA or T4 RNA ligases. In the first step, HH/VS ribozyme cleavage occurs co-transcriptionally, followed by site-specific RNase H cleavage in step II. T4 DNA/RNA ligase based ligation is done in step III. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Chemical shifts for nuclei in RNA from the BMRB data bank

Proton (A), carbon (B) and nitrogen chemical shift ranges (C). The most extensive overlap is seen for the sugar H2′, H3′, H4′, H5′, H5′.

Fig. 7. Typical H1′-H6/H8 assignment ‘walk’ for an RNA thermometer

Spectral assignments were confirmed using the smaller constructs of Fig. 5 which are shown with various colored lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Long-range interactions between the C-terminal tail of Rbfox2 RRM and the lower stem of pre-miR-20b detected by PRE. (A) Overlays of ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled Rbfox2 RRM in complex with paramagnetic spin-labeled pre-miR-20b before (red) and after (green) reduction of the spin label introduced at position U43. Some resonances from the C-terminal tail of the Rbfox2 RRM, which are broadened by the paramagnetic spin label of pre-miR-20b, are annotated. (B) Intensity ratios of NH cross-peaks from the Rbfox2 RRM in complex with pre-miR-20b, between paramagnetic and diamagnetic forms. Residues from the β2β3 loop (around Glu152) and the C-terminal region (Val195–Val215) show significant depressions, indicating long-range contacts between the bottom part of the RNA and the protein. (C) Cartoon representation showing how the highly conserved C-terminal tail of the Rbfox2 RRM can reach the bottom part of the stem-loop to provide additional intermolecular interactions. The figure is adapted from the supplementary information in Ref. [114]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 9. NMR spectroscopy of large RNA Structures (> 65 nt)

RNA structures determined using NMR spectroscopy in last fifteen years are shown here, from the 70 nts thermometer to the 155 nts conserved retroviral RNA packaging element.