key: cord-0275139-69dh2mkp authors: Chen, Silian; Rong, Miao; Lv, Yun; Zhu, Deyu; Xiang, Ye title: Regulation of cGAS activity through RNA-mediated phase separation date: 2020-09-28 journal: bioRxiv DOI: 10.1101/2020.09.27.316166 sha: a4e979003ce0e926357a81f8661a700a86063e73 doc_id: 275139 cord_uid: 69dh2mkp Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA (dsDNA) sensor that functions in the innate immune system. Upon binding dsDNA in the cytoplasm, cGAS and dsDNA form phase-separated aggregates in which cGAS catalyzes synthesis of 2’3’-cyclic GMP-AMP that subsequently triggers a STING-dependent, type I IFN response. Here, we showed that cytoplasmic RNAs, especially tRNAs, regulate cGAS activity. We discovered that RNAs did not activate cGAS but rather promoted phase separation in vitro. In cells, cGAS colocalized with RNAs and formed phase-separated granules even in the absence of cytoplasmic dsDNA. An Opti-prep gradient analysis of cell lysates showed that the endogenous cGAS was associated with cytoplasmic RNAs in an aggregative form. Further in vitro assays showed that RNAs compete for binding of cGAS with dsDNA and inhibit cGAS activity when the dsDNA concentration is high and promote the formation of phase separations and enhance cGAS activity when the dsDNA concentration is low. Thus, cytoplasmic RNAs regulate cGAS activity by interfering with formation of cGAS-containing aggregates. Recognition of pathogen-derived nucleic acids by protein sensors allows the 43 innate immune system to sense infection and initiate host defense mechanisms (1-3). 44 The cyclic GMP-AMP synthase (cGAS) is the primary cytosolic double-stranded 45 DNA (dsDNA) sensor in mammalian cells (4-7). Upon binding to dsDNA, cGAS 46 undergoes conformational changes that activate its ability to catalyze synthesis of a 47 noncanonical 2'3' cyclic-GMP-AMP dinucleotide (2'3'-cGAMP) that triggers type I 48 interferon production through the endoplasmic reticulum membrane protein STING 49 (also known as TMEM173, MPYS, MITA, and ERIS) (8-13). cGAS binds dsDNA in 50 a sequence-independent manner (14-20). Abnormal activity of cGAS can lead to 51 disease such as Aicardi-Goutières syndrome (21). cGAS activity is regulated by 52 degradation or modification of the enzyme through ubiquitination, SUMOylation, 53 phosphorylation, or glutamylation (22-25). A recent report suggests that cGAS 54 activity is also regulated by the formation of phase-separated aggregates upon 55 dsDNA engagement, which confine the activated cGAS to a particular location (26). 56 Previous studies indicated that a variety of parameters, including the concentration 57 and length of the dsDNA, influence sensitivity of cGAS-mediated detection of 58 cytosolic DNA (27, 28). However, cellular cGAS activity is not well explained by 59 current structural and biophysical models (26, 28). 60 Here we showed that cGAS activity is regulated through RNA-mediated phase 61 separation. We found that cGAS forms phase-separated granules with RNAs as well 62 as dsDNA. Aggregation with cytoplasmic RNAs, especially tRNAs, promotes enzyme Figure S1C ). Short dsDNAs with one or more ssDNA arms 77 induced strong phase separations of FL-hcGAS and activated the enzyme ( Figure S2 ). 78 We next performed similar assays using total RNA extracted from HeLa cells. The interactions between cGAS and RNAs in a cytoplasmic extract of Hela 107 6 cells were analyzed using an Opti-prep gradient ( Figure 2B ). There were five major 108 bands in the gradient after centrifugation ( Figure 2C ). hcGAS was detected in 109 fractions from each of these bands by western blot with a cGAS-specific antibody. The endogenous cGAS proteins was located mainly in band 5. Band 5 was sensitive To verify this, we performed in vitro assays with the 129 7 fluorescein-5-thiosemicarbazide-labeled tRNA (FTSC-tRNA). Different amounts of 130 ISD were added to solutions containing the preformed FTSC-tRNA-cGAS granules, 131 and then the granules were separated from the solution by centrifugation and the 132 signal due to FTSC-tRNA was measured in the supernatants ( Figure 3A ). As the 133 concentration of the dsDNA was increased, the FTSC-tRNA signal increased in the 134 supernatant until a plateau was reached ( Figure 3B ). This is indicative of a gradual 135 substitution of the tRNAs by the dsDNAs until a dynamic equilibration was reached. Similarly, when granules were preformed from ISD and hcGAS, we observed a 137 concentration dependent substitution of the dsDNAs by tRNAs ( Figure 3C Nucleic acid recognition by the innate 511 immune system Microbial sensing by Toll-like receptors and intracellular nucleic 513 acid sensors Innate immune sensing and signaling of cytosolic nucleic acids Cyclic GMP-AMP synthase is a cytosolic DNA sensor 517 that activates the type I interferon pathway Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling 519 by cytosolic DNA Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant 521 effects Cytosolic DNA Sensing: The Field Narrows Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous 524 high-affinity ligand for STING Structure-function analysis of STING activation by c[G(2',5')pA(3',5')p] and 526 targeting by antiviral DMXAA STING is an endoplasmic reticulum adaptor that facilitates innate 528 immune signalling The adaptor protein MITA links virus-sensing receptors to IRF3 transcription 530 factor activation MPYS, a novel membrane tetraspanner, is associated with major 532 histocompatibility complex class II and mediates transduction of apoptotic signals ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune 535 signaling through dimerization cGAS is essential for cellular senescence Apoptotic caspases prevent the induction of type I interferons by 539 mitochondrial DNA Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN 541 production Mitochondrial DNA stress primes the antiviral innate immune response. 543 DNA tumor virus oncogenes antagonize the 545 cGAS-STING DNA-sensing pathway cGAS surveillance of micronuclei links genome instability to innate 547 immunity cGAS is essential for the antitumor effect of immune checkpoint blockade Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune 551 diseases The E3 ubiquitin ligase RNF185 facilitates the cGAS-mediated innate immune 553 response Sumoylation Promotes the Stability of the DNA Sensor cGAS and the Adaptor 555 STING to Regulate the Kinetics of Response to DNA Virus Akt Kinase-Mediated Checkpoint of cGAS DNA Sensing Pathway Glutamylation of the DNA sensor cGAS regulates its binding and synthase activity 559 in antiviral immunity DNA-induced liquid phase condensation of cGAS activates innate immune 561 signaling cGAS is activated by DNA in a length-dependent manner cGAS senses long and HMGB/TFAM-bound U-turn DNA by forming 565 protein-DNA ladders Structural mechanism of cytosolic DNA sensing by cGAS cGAS Reveals a Conserved Family of Second-Messenger Enzymes in Innate Immunity Cytosolic RNA:DNA hybrids activate the cGAS-STING axis Intracellular water exchange for measuring the dry mass, water mass 574 and changes in chemical composition of living cells Non-coding RNA: what is functional and what is junk? 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