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Updated: May 7, 2025

Nanomanipulation of Single RNA Molecules by Optical Tweezers
Published on: August 20, 2014
Spontaneous base flipping helps drive Nsp15's preferences in double stranded RNA substrates.
Zoe M Wright1, Kevin John Butay2,3, Juno M Krahn2
1Molecular and Cellular Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, 111 T. W. Alexander Drive, Research Triangle Park, NC, 27709, USA. zoe.wright@nih.gov.
Coronaviruses use Nsp15 endoribonuclease to evade immune detection by cleaving viral RNA. This study shows Nsp15 preferentially cleaves accessible, unpaired uridines, aiding viral immune evasion strategies.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Coronaviruses employ the Nsp15 endoribonuclease to evade host immune surveillance.
- Nsp15 regulates viral double-stranded RNA levels by cleaving uridine bases.
- The mechanism by which Nsp15 targets uridines, specifically whether it induces or utilizes base flipping, remains unclear.
Purpose of the Study:
- To investigate the role of uridine base flipping in Nsp15-mediated RNA cleavage.
- To correlate sequence context, base flipping propensity, and Nsp15 cleavage efficiency.
- To elucidate the mechanistic basis of Nsp15's substrate recognition.
Main Methods:
- Design and synthesis of fluorinated double-stranded RNA substrates.
- Nuclease activity assays.
- Fluorine-19 Nuclear Magnetic Resonance (19F NMR) spectroscopy.
- Mass spectrometry.
- Single particle cryo-electron microscopy (cryo-EM).
Main Results:
- Nsp15 demonstrates highest cleavage efficiency on unpaired and spontaneously flipped uridines.
- Cleavage efficiency is directly proportional to the tendency of uridine bases to flip out of the RNA helix.
- Nsp15 activity is influenced by the sequence context surrounding the target uridine.
- Structural data revealed Nsp15 preferentially binds and cleaves accessible, unpaired uridines.
Conclusions:
- Nsp15's substrate preference is linked to the inherent dynamics of uridine bases within the RNA structure.
- The enzyme likely capitalizes on pre-existing base-flipped or bulged uridines in viral RNA.
- These findings provide a unified understanding of Nsp15 cleavage preferences and its role in coronavirus immune evasion.
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