Characterization of a novel multiprotein complex downstream of the cytosolic nucleic acid sensors cGAS and RIG-I

Abstract

Detection of viral nucleic acids by specialized PRRs of the innate immune system triggers the expression of type I IFNs, a hallmark of antiviral defense. In vertebrates, the cGAS-STING pathway and the RLR-MAVS pathway have emerged as the major cytosolic type I IFN-inducing nucleic acid sensing systems. Although both systems sense the genetic material of different virus classes, signaling downstream of the initial receptor-ligand contact involves common kinases (i.e., TBK1, IKK&#949;) and transcription factors (i.e., IRF3, NF-&#954;B p65). <br /> This thesis has characterized the role of hnRNPM, a predominantly nuclear protein, in cytosolic nucleic acid sensing in the monocytic cell line THP-1. hnRNPM positively regulated both the cGAS-STING- and RIG-I-dependent type I IFN induction by promoting the phosphorylation of TBK1 and/or IRF3. A combined AP-MS/RNAi screen identified ELAVL1 and SON as hnRNPM interactors that also promote cGAS and RIG-I signaling by enhancing TBK1 and/or IRF3 phosphorylation. IGF2BP2, an RNA-dependent hnRNPM interactor, amplified the cGAS- and RIG-I-induced type I IFN response in a TBK1 phosphorylation-independent manner. <br /> To further evaluate the function of ELAVL1, the <em>ELAVL1</em> gene locus was deleted in THP-1 cells. A more detailed investigation revealed that ELAVL1 promoted both the expression of type I IFNs and activation of NF-&#954;B after stimulation of cGAS or RIG-I. The type I IFN response was downregulated by approximately 6-fold to 8-fold in ELAVL1 KO cells and was dependent on the N- and C-terminal RRMs of ELAVL1. Overexpression of ELAVL1-FLAG in ELAVL1-deficient THP-1 dual cells rescued the cGAS- or RIG-I-induced phosphorylation of TBK1/IRF3 and the subsequent ISG response. By contrast, the TLR1/TLR2-mediated phosphorylation of TBK1 was ELAVL1-independent, indicating that ELAVL1 might regulate the activation of different TBK1 subpopulations in a PRR-dependent manner. Analyzing the global mRNA expression profile further demonstrated that ELAVL1 does not regulate the expression of genes associated with cGAS and RIG-I signaling. <br /> A comparative interactome analysis identified common interactors of hnRNPM and ELAVL1, including RNA-binding proteins, ribosomal proteins, splicing factors, and also type I IFN-inducing proteins (i.e., DDX3X). The interactions with SON were restricted to hnRNPM, suggesting that hnRNPM forms distinct complexes with ELAVL1 and SON. In addition, hnRNPM also interacted with TBK1, IKK&#949;, IKK&#946;, and NF-&#954;B p65. PLAs confirmed that the interactions between hnRNPM, ELAVL1, and TBK1 phosphorylated at Ser172 occur <em>in cellulo</em> and primarily in the cytoplasm, indicating that the minor cytoplasmic rather than the predominant nuclear portions of hnRNPM and ELAVL1 mediate TBK1 phosphorylation. Furthermore, the antibody-based purification of the multiprotein complex assembled by hnRNPM enhanced the phosphorylation of recombinant TBK1 in the activation loop. <br /> In summary, this thesis provides new insights into the signaling cascades downstream of cGAS and RIG-I and has identified a novel multiprotein complex that positively regulates both pathways at a common point of convergence, TBK1 phosphorylation. A better understanding of the newly identified multiprotein complex might contribute to the development of new therapies for the treatment of inflammatory diseases resulting from dysregulation of cGAS-STING or RIG-I-MAVS signaling (i.e., AGS, SMS, or SAVI).