Cargando…

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

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 cy...

Descripción completa

Detalles Bibliográficos
Autores principales: Shah, Neelam, Beckham, Simone A, Wilce, Jacqueline A, Wilce, Matthew C J
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887321/
https://www.ncbi.nlm.nih.gov/pubmed/29346611
http://dx.doi.org/10.1093/nar/gkx1307
_version_ 1783312275529007104
author Shah, Neelam
Beckham, Simone A
Wilce, Jacqueline A
Wilce, Matthew C J
author_facet Shah, Neelam
Beckham, Simone A
Wilce, Jacqueline A
Wilce, Matthew C J
author_sort Shah, Neelam
collection PubMed
description 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-AlF(x) 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.
format Online
Article
Text
id pubmed-5887321
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-58873212018-04-11 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 Nucleic Acids Res RNA and RNA-protein complexes 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-AlF(x) 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. Oxford University Press 2018-04-06 2018-01-13 /pmc/articles/PMC5887321/ /pubmed/29346611 http://dx.doi.org/10.1093/nar/gkx1307 Text en © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle RNA and RNA-protein complexes
Shah, Neelam
Beckham, Simone A
Wilce, Jacqueline A
Wilce, Matthew C J
Combined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis
title Combined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis
title_full Combined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis
title_fullStr Combined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis
title_full_unstemmed Combined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis
title_short Combined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis
title_sort combined roles of atp and small hairpin rna in the activation of rig-i revealed by solution-based analysis
topic RNA and RNA-protein complexes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887321/
https://www.ncbi.nlm.nih.gov/pubmed/29346611
http://dx.doi.org/10.1093/nar/gkx1307
work_keys_str_mv AT shahneelam combinedrolesofatpandsmallhairpinrnaintheactivationofrigirevealedbysolutionbasedanalysis
AT beckhamsimonea combinedrolesofatpandsmallhairpinrnaintheactivationofrigirevealedbysolutionbasedanalysis
AT wilcejacquelinea combinedrolesofatpandsmallhairpinrnaintheactivationofrigirevealedbysolutionbasedanalysis
AT wilcematthewcj combinedrolesofatpandsmallhairpinrnaintheactivationofrigirevealedbysolutionbasedanalysis