Fig. 7: CircRIG-I activates DDX3X-mediated innate immune signaling.
From: Mutant RIG-I enhances cancer-related inflammation through activation of circRIG-I signaling
a Workflow diagram of circRNA-MS2-MCP-TurboID system. MS2 is a stem-loop bacteriophage RNA that specifically binds to the MS2 coating protein (MCP) with high affinity. By fusing MCP and biotin ligase TurboID, adjacent proteins of MS2 tagged circRIG-I are covalently labeled by biotin. Followed by streptavidin beads enrichment of biotinylated proteins and mass spectrometry, circRIG-I-associated proteins can be identified. b Mass spectrum analysis of circRIG-I-associated proteins according to the workflow shown in (a). Data are collected from 2 independent experiments. c Vectors encoding DDX3X-FLAG and murine-derived circRIG-I were co-transfected into HEK293T cells. After 24 hours of transfection, RNA pulldown assay was performed by biotin labeled nucleotide probe specific to circRIG-I. Data are collected from 2 independent experiments. d Confocal examination of the colocalization between DDX3X and circRIG-I in iBMDM cells. The scale bars represent 20 μm. Data are collected from 2 independent experiments. e HEK293T cells were transfected with vectors encoding DDX3X-FLAG or murine circRIG-I. UV-CLIP assay was performed and followed RT-qPCR analysis verified the protein-circRNA association (n = 3 cell cultures, mean ± s.e.m., ns, ****P < 0.0001, two-tailed unpaired Student’s t-test). f Mass spectrum analysis of DDX3X-associated proteins in presence or absence of circRIG-I. The band indicating DDX3X was shown. Data are collected from 2 independent experiments. g Protein-protein interaction (PPI) analysis of DDX3X interactome in presence or absence of circRIG-I by Cytoscape Microsoft. h HEK293T cells were co-transfected with vectors encoding FLAG tagged proteins and DDX3X-GFP with or without murine-derived circRIG-I. At 24 hours later, cell lysates were immunoprecipitated with FLAG antibody and protein A/G agarose beads and analyzed by immunoblot with anti-GFP antibody. Data are collected from 2 independent experiments.The primers used for quantitative real-time PCR have been deposited in Supplementary Data 5. Source data are provided as a Source Data file.
