Combined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis

Research output: Contribution to journalArticleResearchpeer-review

Abstract

RIG-I (retinoic acid inducible gene-I) is a cytosolic innate immune protein that senses viral dsRNA with a 5-triphosphate overhang. Upon interaction with dsRNA a de-repression of the RIG-I CARD domains takes place that ultimately leads to the production of type I interferons and pro-inflammatory cytokines. Here we investigate the RIG-I conformational rearrangement upon interaction with an activating 5-triphosphate-10-base pair dsRNA hairpin loop (10bp) compared with a less active 5-triphosphate-8-base pair dsRNA hairpin loop (8bp). We use size-exclusion chromatography-coupled small-angle X-ray scattering (SAXS) and limited tryptic digest experiments to show that that upon binding to 10 bp, but not 8 bp, RIG-I becomes extended and shows greater flexibility, reflecting the release of its CARDs. We also examined the effect of different ATP analogues on the conformational changes of RIG-I/dsRNA complexes. Of the analogues tested, the addition of ATP transition state analogue ADP-AlFx further assisted in the complete activation of RIG-I in complex with 10bp and also to some extent RIG-I bound to 8bp. Together these data provide solution-based evidence for the molecular mechanism of innate immune signaling by RIG-I as stimulated by short hairpin RNA and ATP.

Original languageEnglish
Pages (from-to)3169-3186
Number of pages18
JournalNucleic Acids Research
Volume46
Issue number6
DOIs
Publication statusPublished - 6 Apr 2018

Cite this

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title = "Combined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis",
abstract = "RIG-I (retinoic acid inducible gene-I) is a cytosolic innate immune protein that senses viral dsRNA with a 5-triphosphate overhang. Upon interaction with dsRNA a de-repression of the RIG-I CARD domains takes place that ultimately leads to the production of type I interferons and pro-inflammatory cytokines. Here we investigate the RIG-I conformational rearrangement upon interaction with an activating 5-triphosphate-10-base pair dsRNA hairpin loop (10bp) compared with a less active 5-triphosphate-8-base pair dsRNA hairpin loop (8bp). We use size-exclusion chromatography-coupled small-angle X-ray scattering (SAXS) and limited tryptic digest experiments to show that that upon binding to 10 bp, but not 8 bp, RIG-I becomes extended and shows greater flexibility, reflecting the release of its CARDs. We also examined the effect of different ATP analogues on the conformational changes of RIG-I/dsRNA complexes. Of the analogues tested, the addition of ATP transition state analogue ADP-AlFx further assisted in the complete activation of RIG-I in complex with 10bp and also to some extent RIG-I bound to 8bp. Together these data provide solution-based evidence for the molecular mechanism of innate immune signaling by RIG-I as stimulated by short hairpin RNA and ATP.",
author = "Neelam Shah and Beckham, {Simone A.} and Wilce, {Jacqueline A.} and Wilce, {Matthew C.J.}",
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Combined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis. / Shah, Neelam; Beckham, Simone A.; Wilce, Jacqueline A.; Wilce, Matthew C.J.

In: Nucleic Acids Research, Vol. 46, No. 6, 06.04.2018, p. 3169-3186.

Research output: Contribution to journalArticleResearchpeer-review

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