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

• Published in: Nucleic acids research 2018-04, Vol.46 (6), p.3169-3186
• Main Authors: Shah, Neelam, Beckham, Simone A, Wilce, Jacqueline A, Wilce, Matthew C J
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Base Sequence; Chromatography, Gel; DEAD Box Protein 58 - chemistry; DEAD Box Protein 58 - metabolism; Humans; Models, Molecular; Nucleic Acid Conformation; Protein Binding; Protein Domains; Receptors, Immunologic; RNA and RNA-protein complexes; RNA, Double-Stranded - chemistry; RNA, Double-Stranded - genetics; RNA, Double-Stranded - metabolism; Scattering, Small Angle; Signal Transduction; Solutions - chemistry; X-Ray Diffraction
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkx1307

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.