Advances that facilitate the study of large RNA structure and dynamics by nuclear magnetic resonance spectroscopy

Primer

Huaqun Zhang, Sarah C. Keane

Published Online: Apr 25 2019

DOI: 10.1002/wrna.1541

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Abstract The characterization of functional yet nonprotein coding (nc) RNAs has expanded the role of RNA in the cell from a passive player in the central dogma of molecular biology to an active regulator of gene expression. The misregulation of ncRNA function has been linked with a variety of diseases and disorders ranging from cancers to neurodegeneration. However, a detailed molecular understanding of how ncRNAs function has been limited; due, in part, to the difficulties associated with obtaining high‐resolution structures of large RNAs. Tertiary structure determination of RNA as a whole is hampered by various technical challenges, all of which are exacerbated as the size of the RNA increases. Namely, RNAs tend to be highly flexible and dynamic molecules, which are difficult to crystallize. Biomolecular nuclear magnetic resonance (NMR) spectroscopy offers a viable alternative to determining the structure of large RNA molecules that do not readily crystallize, but is itself hindered by some technical limitations. Recently, a series of advancements have allowed the biomolecular NMR field to overcome, at least in part, some of these limitations. These advances include improvements in sample preparation strategies as well as methodological improvements. Together, these innovations pave the way for the study of ever larger RNA molecules that have important biological function. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems

Methods for fragmentation or segmental labeling of large RNAs. (a) Secondary structure of a large RNA with a complex multi‐helix junction. (b) “Divide and conquer” approach. Small oligo RNAs are designed to mimic the structure of particular regions within the large RNA. These oligo RNAs are well‐suited for rapid chemical shift assignment by traditional methods. (c) Enzymatic ligation allows for segmental labeling of a large RNAs. Two RNAs are independently synthesized and can therefore be independently labeled. The two RNAs can then be ligated together to make a full‐length construct with only one region of the RNA containing nuclear magnetic resonance‐active isotope labeling. (d) Fragmentation‐based segmental labeling. The RNA of interest is fragmented at a hairpin loop. The loop sequence is replaced (in some cases) with a run of intermolecular C–G base pairs which serve as an annealing handle. This approach in theory can be applied at any hairpin loop structure within a large RNA molecules

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Deuterium labeling greatly improves spectral quality and simplicity. (a) ¹H‐¹H NOESY spectrum of the full‐protiated (H, unlabeled) 155 nucleotides HIV‐1 core encapsidation signal (Keane et al., ) is characterized by severe signal overlap and broad line widths. (b) Selective deuteration of the sample results in spectra of significantly higher quality. There is minimal signal overlap and resonances are now effectively identifiable by the respective labeling scheme. Four different labeling schemes are shown: A^H (adenosines protiated, cytosines, guanosines, and uracils deuterated), C^H (cytosines protiated, adenosines, guanosines, and uracils deuterated), G^H (guanosines protiated, adenosines, cytosines, and uracils deuterated), U^6R (uracils protiated on the ribose and at C₅, adenosines, cytosines, and guanosines deuterated)

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Histogram of RNA structures that were determined using nuclear magnetic resonance spectroscopy. The size of the RNA molecule (nucleotides, nt) is correlated with the number of structures reported in the Protein Data Bank (rcsb.org, June 2018)

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Detection of hydrogen bonds in canonical A–U and G–C base pairs. Traditionally, imino protons (blue) are used to infer base pairing within helical structural elements. Improvements to pulse sequences, implemented by the Sattler lab, allow for the detection of hydrogen‐bonding on nonexchangeable C₂ (adenosine) and C₅ (cytosine and uracil) protons (Dallmann et al., ). Magnetization transfer pathways are indicated with arrows

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