Cellular RNA interacts with MAVS to promote antiviral signaling

Immune signaling needs to be well-regulated to promote clearance of pathogens, while preventing aberrant inflammation. Interferons (IFNs) and antiviral genes are activated by the detection of viral RNA by RIG-I-like receptors (RLRs). Signal transduction downstream of RLRs proceeds through a multiprotein complex organized around the central adaptor protein MAVS. While protein-protein interactions and post-translational modifications are critical for the formation of the MAVS signalosome, whether RNA play a role in organizing these platforms is largely unknown. RNA can modulate protein complex function by allosteric regulation or by serving as a molecular guide or scaffold. We have found that MAVS directly interacts with the 3’ untranslated regions of cellular mRNAs through its central intrinsically disordered domain. Eliminating RNA by RNase treatment disrupts the MAVS signalosome and inhibits phosphorylation of the transcription factor IRF3. These findings support the hypothesis that RNA molecules scaffold the MAVS signaling platform to induce IFNs. Indeed, RNase treatment alters interactions between MAVS and newly identified regulators of RLR signaling. Together, this work uncovers a function for cellular RNA in promoting signaling through MAVS and highlights a generalizable principle of RNA regulatory control of cytoplasmic immune signaling complexes. We used targeted RNA editing mediated by APOBEC1 to identify MAVS-associated RNAs. Targeted APOBEC1-mediated editing can robustly identify the RNA interactomes of RBPs without relying on crosslinking or immunoprecipitation. Here, APOBEC1 fused to the protein of interest catalyzes C-to-U transitions in RNAs that interact with this protein, which can be detected as C-to-T mutations by RNA sequencing (Brannan et al. 2021; Flamand et al. 2022). To profile MAVS-associated RNAs, we fused FLAG-tagged APOBEC1 to the N-terminus of full-length MAVS (APOBEC1-MAVSFL). We used FLAG-APOBEC1 as a background control (CTRL), and FLAG-APOBEC1 fused to the C-terminal transmembrane helix of MAVS (MAVSC-term) as a mitochondrially-localized background control to account for off-target editing within the subcellular vicinity of MAVS. RNA-sequencing data from 293T cells transfected with these constructs were analyzed using the Bullseye pipeline to identify RNAs with increased C-to-T editing with APOBEC1-MAVSFL as compared to controls (https://github.com/mflamand/Bullseye; Flamand et al. 2022; Tegowski et al. 2022). C-to-T editing was detected at sites with at least 10 reads of coverage per sample, with an edit ratio between 2% and 95% (minEdit=5), and an edit ratio at least 1.5-fold higher than control samples (editFoldThreshold=1.5)