CCR5 RNA Pseudoknots: Residue and Site‐Specific Labeling correlate Internal Motions with microRNA Binding

• Published in: Chemistry : a European journal 2018-04, Vol.24 (21), p.5462-5468
• Main Authors: Chen, Bin, Longhini, Andrew P., Nußbaumer, Felix, Kreutz, Christoph, Dinman, Jonathan D., Dayie, T. Kwaku
• Format: Article
• Language: English
• Subjects: Binding; CCR5 mRNA pseudoknot; CCR5 protein; Chemistry; Couplings; DNA probes; dynamics; Labeling; Line broadening; microRNA; MicroRNAs; MicroRNAs - chemistry; MicroRNAs - metabolism; miRNA; Models, Molecular; NMR; NMR spectroscopy; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular; Nucleotides; Perturbation methods; Receptors, CCR5 - genetics; Ribonucleic acid; RNA; RNA probes; RNA solid-phase synthesis; Solid phase synthesis; Solid-Phase Synthesis Techniques; Stimulators; Structure-function relationships
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.201705948

Conformational dynamics of RNA molecules play a critical role in governing their biological functions. Measurements of RNA dynamic behavior sheds important light on sites that interact with their binding partners or cellular stimulators. However, such measurements using solution‐state NMR are difficult for large RNA molecules (>70 nt; nt=nucleotides) owing to severe spectral overlap, homonuclear 13C scalar couplings, and line broadening. Herein, a strategic combination of solid‐phase synthesis, site‐specific isotopic labeled phosphoramidites, and enzymatic ligation is introduced. This approach allowed the position‐specific insertion of isotopic probes into a 96 nt CCR5 RNA fragment. Accurate measurements of functional dynamics using the Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments enabled extraction of the exchange rates and populations of this RNA. NMR chemical shift perturbation analysis of the RNA/microRNA‐1224 complex indicated that A90‐C1′ of the pseudoknot exhibits similar changes in chemical shift observed in the excited state. This work demonstrates the general applicability of a NMR‐labeling strategy to probe functional RNA structural dynamics. Genes in a twist: A highly conserved pseudoknot element within the mRNA encoding human C‐C chemokine receptor type 5 self‐regulates gene expression through a translational recoding event. A residue/site‐specific labeling strategy was applied to insert isotopic probes into the pseudoknot. This approach enables unambiguous assignments of key residues involved in intermolecular interaction with miRNA‐1224 and results in accurate measurements of their dynamics at atomic resolution.