TRIM25-mediated RIG-I signaling is one of the key steps in the host response against a broad spectrum of RNA viruses, many of which pose a significant hazard for public health and human wellbeing, such as Influenza, Dengue, Ebola and the novel coronaviruses (1) . Many of these viruses have developed host-pathogen interactions to evade host immunity by interfering with TRIM25-mediated RIG-I ubiquitination (2) (3) (4) (5) (6) (7) . The recent outbreaks of emerging human pathogens such as Ebola in Western Africa in 2013-2016 or the ongoing SARS-CoV-2 pandemic, the steady rise in Dengue virus infections over the last 50 years as a consequence of human-made climate change, as well as the seasonal death toll caused by Influenza virus, which could be significantly increased in the event of a pandemic, highlight the importance of understanding these host-pathogen interactions as a potential target for fighting these diseases.
In this study we focus on the role of RNA binding of the E3 ligase TRIM25 in its physiological functions and its exploitation by viruses in host immunity evasion (8) (9) (10) (11) . TRIM25 is part of the tripartite motif (TRIM) family of ubiquitin ligases characterized by an N-terminal RING domain, followed by one or two B-box domains, a coiled-coil (CC) dimerization domain and a C-terminal region, which, depending on the subfamily, can feature various domains ( Figure 1A ). TRIM25 is a member of the PRY/SPRY domain subfamily and the first member of this subgroup where RNA binding has been observed (6, (12) (13) (14) . TRIM25 has reported functions in innate immunity, morphogenesis and cell proliferation (15) (16) (17) (18) (19) , but mechanistic understanding of its mode of action is scarce.
The best characterized function of TRIM25 is its role in RIG-I signalling during the antiviral response.
Here, it ubiquitinates the RIG-I caspase activation and recruitment domains (CARDs), which are exposed upon recognition of 5′ triphosphate blunt-end double-stranded RNA (15, (20) (21) (22) . The importance of this pathway in innate immunity is highlighted by the variety of mechanisms that viruses have evolved to inhibit TRIM25 activity and evade the host defense (2) (3) (4) (5) (6) (7) . TRIM25 has been shown to also ubiquitinate other targets, often RNA-binding proteins. In embryonic stem cells, TRIM25 is required for the binding of Lin28a to the precursor of let-7 (pre-let-7), which in turn recruits Tut4 to the RNA (23) . Tut4, after activation through ubiquitination by TRIM25, poly-uridylates pre-let-7 and thereby marks it for degradation. Also, TRIM25 ubiquitinates the zinc-finger antiviral protein (ZAP), a protein interacting with viral mRNA and facilitating exosome-mediated degradation (24) . Various RNAs seem to act as co-factors for TRIM25's E3 ubiquitin ligase activity, facilitating autoubiquitination and ubiquitination of RIG-I and ZAP (25) (26) (27) . While most studies point towards an activating role of RNA on TRIM25 in vitro, there is some evidence that the Dengue virus uses its subgenomic RNA to inhibit TRIM25 and its role in interferon expression (3) .
Despite clear evidence for the importance of RNA-binding for TRIM25 activity, the molecular mechanism of this activation remains unexplained and structural data on the TRIM25-RNA interaction is lacking. The identity of the RNA-binding domain also remains controversial, with the CC domain originally being identified (12) , while more recent data point towards the PRY/SPRY domain (25) . In addition, a lysine-rich part of the linker connecting CC and PRY/SPRY domain was proposed to be involved in this interaction (26) . Here, we resolve this controversy by showing that the CC and PRY/SPRY domains act together cooperatively to bind RNA. We have previously shown that the PRY/SPRY and CC interact weakly in solution (28) and that this interaction is necessary for RIG-I ubiquitination. Binding to RNA enhances the CC-PRY/SPRY interaction, mechanistically explaining the observed activation of TRIM25 E3-ubiquitin ligase activity. Functional assays in mammalian cells confirm the importance of RNA binding interfaces on both domains, as RNA binding mutants lessen RIG-I ubiquitination. RNA-binding also induces the formation of macromolecular condensates (liquidliquid phase separation) in vitro and recruits RIG-I CARDs into these condensates. TRIM25 also forms condensates in cells. This suggests a completely new molecular mechanism of TRIM25/RIG-I interaction in which a common RNA acting as a scaffold rather than direct protein/protein-interactions is critical to bring the two proteins together.
TRIM25 CC (aa 189-379), PRY/SPRY (439-630) and CC-PRY/SPRY (189-630) were cloned using restriction free cloning into pETM22 featuring a 3C-protease cleavable hexa-histidine and thioredoxin tag (His-Trx). An alternative N-terminally extended PRY/SPRY construct (407-630) was cloned into pETM20 with a TEV-protease cleavable His-Trx tag. TRIM25 189-379 was expressed in BL21(DE3).
TRIM25 439-630, 407-630 and 189-630 were co-expressed with chaperones KJE, ClpB and GroELS in E. coli BL21(DE3) (29) . All proteins were induced at OD600=0.6 by 200 µM isopropyl-ß-D-1thiogalactopyranoside (IPTG) and incubated at 18˚ C for 20h.
All proteins were purified by immobilized Nickel affinity chromatography (GE Histrap FF) in 50 mM Tris, pH 7.5, 300 mM NaCl and 0.2 mM TCEP and eluted with a gradient of imidazole (10-300 mM removed. Data analysis was done using the ATSAS package version 2.8.3 (38) . PRIMUS was used for frame averaging and buffer subtraction (39) . The radius of gyration, R g , was estimated using the Guinier approximation in PRIMUS. Pair-wise distribution functions were calculated using GNOM (40) .
Collection statistics for SAXS measurements are summarized in Supplementary Table S1 .
Filter binding assays were carried out in 200 µl of binding buffer (20 mM MES, pH 6.5, 75 mM NaCl, 0.5 mM TCEP). Structured RNA probes were refolded prior to experiments by heating at 85°C for 3 min and slow cooling to room temperature. A concentration series of TRIM25 CC-PRY/SPRY was incubated with 5′ 32 P-labelled RNA for 10 min on ice and the sample were filtered through Whatman 0.45μm nitrocellulose filters. The protein/RNA complex was retained on the filters and detected by scintillation counting. Binding curves were fitted using SciDavis as:
Where A max and A ሺ ܿ ௧ ሻ are measured activities. This approach only gives valid results, if the K D is much smaller than the concentration of radio-labelled RNA. In cases, were this assumption was false the K D was corrected as follows:
In cell ubiquitination of RIG-I CARDs by TRIM25 and its mutants was assessed as previously described (28) . In short, HEK293T cells were cultured in DMEM with 10% FBS and antibiotics and seeded in 6 well plates (5x10 5 cells per well) 24 hours prior transfection. Cells were transfected using 
HeLa cells were cultured in DMEM with 10% FBS and antibiotics and seeded in glass bottom plates (3x10 5 cells per well) 24 hours prior transfection. Cells were transfected using Fugene transfection reagent and the respective expression constructs for mEGFP-TRIM25 and dTomato-RIG-I CARDs (aa 2-200) in pcDNA3. Oxidative stress was induced by treatment with 0.5 mM sodium (meta)arsenite for 2 hours. Cells were imaged at 37 ˚C on an Olympus FV3000 inverted confocal microscope equipped with an Olympus UPL SAPO 40x2 NA 0.95 objective.
TRIM25 CC-PRY/SPRY (WT or K283A/K285A/H505E/K508E/K602E pentamutant) and DENV-SL or pre-let-7 were mixed in equimolar ratios at concentrations between 1 and 10 µM in 20 mM sodium phosphate, pH 6.5, 100 mM NaCl and 0.5 mM TCEP at room temperature and immediately imaged using differential interference contrast (DIC) on an Olympus FV3000 inverted confocal microscope and Olympus UPL SAPO 40x2 NA 0.95 objective. To assess localisation of RIG-I with TRIM25/DENV-SL, bacterially expressed dTomato-RIG CARDs or dTomato alone were added at 3 µM to macromolecular condensates pre-formed at 5 µM and distribution of dTomato was observed by confocal fluorescence microscopy. Fluorescence intensity as a measure of protein concentration in the droplets and surrounding media was quantified using Fiji (41) . For each condition fluorescence intensity was measured for at least twenty droplets and an equivalent number of points in the surrounding media distributed over at least three frames, normalized by the average intensity of the frame, averaged and plotted using SciDavis.
Since RNA-binding of TRIM25 is structurally uncharacterized, we performed NMR titrations with the TRIM25 PRY/SPRY domain and the reported RNA target pre-let-7a-1 ( Figure 1B) . A minimal pre-let-7a-1@2 construct (pre-let-7 in the following) has been described to promote TRIM25-mediated regulation of let-7 by Lin28a and TuT4 (23) . Upon addition of RNA we observed strong chemical shift perturbations (CSPs) clustered around two regions of the PRY/SPRY domain ( Figure 1B -D). The first of the two binding sites (binding-site-1 in the following) is located at the C-terminus of the PRY motif with the strongest affected residues in the flexible loop connecting ß-strands 3 and 4 (Supplementary Figure S1 ). This site is located in close proximity to the interaction site between the PRY/SPRY and CC reported in Koliopoulos et al. (28) . Our data thereby confirm the RNA-binding region (aa 470-508)
reported by Choudhury et al. (25) , but increase the accuracy to residue resolution. The second binding site is located in a region formed by ß-strands 10 and 11 and is located close to the N- Figure S1 ). Although this region was not shown earlier to be involved in RNA binding, a central role in the recruitment of RIG-I has been reported (42) . At this stage, we could not assess, whether the CSPs are due to direct interaction with RNA or due to an allosteric effect.
To further assess RNA specificity of these binding sites we designed shorter RNA constructs consisting of only the loop or stem of pre-let-7 (Supplementary Figure S2 ). Upon titration with the single stranded part of pre-let-7 (pre-let-7 loop), we observed CSPs only for residues located at binding-site-1, whereas binding-site-2 was unaffected ( Figure 1E ). To ensure that the short stem is double-stranded, we fused the strands together by a three bases long linker (pre-let-7 stem). Titration with the resulting RNA construct strongly affected both binding sites, 1 and 2 ( Figure 1F ). Together this suggests that the second binding site is specific for double-stranded RNA, while the first binding site seemingly binds to single-and double-stranded RNA. The presence of two binding sites with distinct binding preferences in close proximity suggests a possible binding selectivity for structural elements rather than sequence specificity. Indeed, comparing other reported RNA targets of TRIM25, we found that all feature stem loops of similar size and sequence as pre-let-7. We used ITC to measure the affinity of several of these stem loops to TRIM25 CC-PRY/SPRY (Table 1) . We observed very tight binding of this construct to pre-let-7 (K D = 72 ± 33nM, Figure 2A ) and similar stem loops Figure S3A ). Due to these complications we could only quantify and compare the highest affinity binding event. The significance of the lower affinity binding events is not clear, but since they generally feature low N-values, they may represent RNA-induced oligomerisation. This is supported by the observation of protein aggregation upon RNA addition for most of these RNAs.
Filter-binding assays confirmed the stronger binding of DENV-SL compared to pre-let-7 and showed binding with nanomolar affinity also for a stem loop of Drosophila lncRNA roX2 ( Table 2 ). The latter is not a reported TRIM25 target but involved in Drosophila dosage compensation (43) and could therefore be considered structurally and functionally unrelated, further supporting that TRIM25 binds a variety of stem-loop RNAs regardless of sequence. Affinities measured by filter-binding assays were generally higher than those by ITC, possibly because filter binding is more sensitive to aggregation.
As reported previously, we found that the affinity of the PRY/SPRY domain for pre-let-7 RNA (5.0±1.2 µM, as measured by ITC, Table 2 , Figure 2B ) is much weaker than for CC-PRY/SPRY (25, 26) . The RNA-binding of the PRY/SPRY domain alone is therefore not sufficient to explain the high affinity binding of TRIM25. The complex binding isotherm suggests that an additional binding site might be necessary for high affinity binding (Supplementary Figure S3A) . This is also supported by the Figure   3L ) than the original study described for binding of double stranded RNA, making it unlikely that this binding site alone is responsible for the observed increase in affinity.
We noted that in the crystal structure of the CC-PRY/SPRY dimer (PDB:6FLN) binding-site-1 is part of a larger positively charged surface that extends onto the CC ( Figure 2B) indicating that the complex adopts a more compact conformation than the free protein in solution ( Figure 2C) , where the PRY/SPRY domain is mostly detached from the CC domain (28) . This is also evident from the pairwise distance distribution, P(r), obtained from the SAXS data, where the free protein shows a broad distribution with two distinct maxima, indicating that the two domains tumble independently. In contrast, the complex shows a single maximum with a much narrower distribution, indicating that it tumbles as a single, compact entity ( Figure 2C ). We conclude that RNA binding enhances the interaction between the CC and PRY/SPRY domains and leads to a more compact conformation of the protein.
To better understand the relative contributions of the PRY/SPRY RNA binding sites identified by NMR, and the binding site on the CC, inferred from the crystal structure of CC-PRY/SPRY ( Figure 2B ), we created point mutants and tested their effect on RNA binding using ITC (Table 1 triple-mutant) further reduces RNA-binding more than ten-fold compared to wildtype (K D = 790 ± 160 nM). This suggests that both binding sites on the PRY/SPRY domain are critical for cooperative binding. The effect of these mutants is however not as strong as that of previously published, less conservative deletions or mutating entire regions (25, 26) .
The design of CC mutants turned out to be more difficult as we could only rely on indirect information in the absence of NMR data and thus inferred RNA binding residues from amino acids which are surface-exposed, close to the PRY/SPRY-CC interface and belong to typical RNA-binding residues like lysines, arginines and aromatic residues. We found that a double mutant on the basic surface close to the PRY/SPRY binding site (K283A/K285A) in the context of the CC-PRY/SPRY reduced RNA-binding about 8-fold (K D = 606 ± 124 nM, Figure 2D ). This surface patch also harbours tyrosine 278, whose phosphorylation was previously shown to affect TRIM25's E3 ligase activity (45) . Since the close proximity to the RNA binding residues might suggest that phosphorylation of Y278 could also affect RNA binding, we tested RNA binding of the Y278A mutant in ITC, but found no effect on binding affinity to pre-let-7. It is however likely that additional residues on the CC are involved in RNA Figure 3B ). It is noteworthy that the effect of both wildtype and mutants is strongest for di-and tri-ubiquitination, while mono-ubiquitination is almost unaffected. It is possible that mono-ubiquitination of CARDs is caused by an E3 ligase other than TRIM25 or RNA binding might be required for chain elongation, but not initiation. The latter could be an E2 specific effect, supported by the observation reported earlier that Ubc13/Uev1A, the E2 complex specific for the production of K63-linked ubiquitin chains in vitro only produces unanchored ubiquitin chains, suggesting that a second E2 is necessary for chain initiation (46, 47) .
This effect is even more striking, given that mutants H505E/K508E and K602E on the PRY/SPRY, but not K283/285A on the CC, showed consistently elevated expression levels compared to the wildtype.
Carfilzomib stabilized both wildtype and H505E/K508E/K602E triple mutant TRIM25, but had a much more dramatic effect on the wildtype, leading to similar expression levels than the mutant ( Figure 3C ).
This points towards a crucial role of TRIM25-RNA binding not only in RIG-I ubiquitination but also in auto-ubiquitination of TRIM25. While in RIG-I ubiquitination TRIM25 specifically produces K63-linked chains, that do not promote proteasomal degradation, for other targets such as MAVS, ZAP or 14-3-3σ degradative K48-linked ubiquitination by TRIM25 was reported and is therefore possibly also occurring during auto-ubiquitination (16, 48, 49) . As the RNA binding deficient mutants include the replacement of lysines, it is possible that this removes the auto-ubiquitination target lysine of TRIM25.
Together with the strong reduction in substrate ubiquitination, this suggests that RNA binding enhances auto-ubiquitination of TRIM25 in cells and thereby regulates its proteasomal degradation and protein levels.
To gain further insight into the structurally so far uncharacterized interaction of TRIM25 and RIG-I, we for their direct interaction in vitro (26) . Several additional factors thought to stabilize the interaction, including both proteins and RNAs, have been proposed (27, 44, 50) .
Since we observed a significant overlap of mutants previously described to reduce TRIM25/RIG-I interaction (42) with our binding-site-2, that is specific to double-stranded RNA, we set out to further investigate the possible stabilization of the TRIM25/RIG-I interaction by RNA. Figure S4) .
Upon induction of stress using sodium arsenite, TRIM25 relocalized to smaller, well defined puncta in the cytosol that in a few cases also contained RIG-I CARDs. This confirms previous research showing that both TRIM25 and RIG-I are constituents of stress granules, a membrane-less organelle formed by liquid-liquid phase separation around mRNAs (26, (51) (52) (53) . In comparison, the H505E/K508/K602E
RNA-binding deficient mutant showed a stronger localization to granules in the nucleus and its vicinity.
It is however not clear if this re-localization is a consequence of reduced RNA-binding, autoubiquitination or increased expression levels of this mutant. Nuclear TRIM25 has been previously reported, but its significance remains unclear (54) . In summary, this shows that TRIM25 can be part of various cytoplasmic and nuclear granules, although this commonly requires a stimulus such as oxidative stress.
Interestingly 20-fold in these droplets, while only less than two-fold enrichment of RFP alone was observed ( Figure   5B ). This is remarkable, since CARDs have no known RNA-binding activity (27, 55) . To our knowledge this is the first evidence for an interaction between TRIM25 and RIG-I in vitro, although it is at this stage not clear if this is due to direct protein/protein interactions or so far unobserved RNA-binding of RIG-I CARDs. However, our NMR experiments rule out a direct PRY/SPRY:RIG-I CARDs interaction unless RNA binding of the PRY/SPRY allosterically increases affinity to CARDs. The observation that the T55I mutation on the CARDs, that was reported to reduce TRIM25/RIG-I interaction in cells, has no such effect in our in vitro assays further shows that a binary PRY/SPRY:CARDs interaction is insufficient to explain the association (2) . Irrespective of the nature of this interaction, this experiment demonstrates that RNA-induced liquid-liquid phase separation could account for the efficient recruitment of RIG-I to TRIM25 despite the absence of detectable interactions between the isolated proteins. The described system might therefore act as a model system to further study the determinants of liquid-liquid phase separation and RIG-I activation by TRIM25.
In summary, we show that TRIM25 achieves very tight binding of RNA through several binding sites on the CC and PRY/SPRY domain, with each binding site showing only weak affinities to RNA and different specificities. While we confirm and refine a previously identified binding region on the PRY/SPRY, we found additional binding sites on the PRY/SPRY and CC. The novel second binding site on the PRY/SPRY seems to be specific for double-stranded RNA and overlaps with a region previously thought to be involved in RIG-I binding (42) . The close proximity of binding sites specific for single and double stranded RNA suggests a specificity for structure rather than sequence and indeed we found binding with nanomolar affinity to several stem-loops regardless of sequence. This may explain the failure of previous studies to identify a clear RNA motif for TRIM25 (23) . The highly cooperative binding mode involving multiple binding sites on different domains is critical for the E3ubiquitin ligase activity of TRIM25, as RNA binding stabilizes the weak interaction between the CC and PRY/SPRY domain which was previously shown to be critical for TRIM25's catalytic activity (28) .
In the absence of structural data of the complete tripartite motif of TRIM25 the importance of this with the finding of us and others, that TRIM25 RNA-binding is required for efficient ubiquitination of the RIG-I CARDs (26) . It is however not clear how RING dimerization, that is necessary for the E3 ligase activity of TRIM25 but not TRIM28, is achieved in this model (46, 47, 58) . Such RING dimerization could occur between two TRIM25 dimers stacking up end to end. Such an inter-dimer association could also be facilitated by RNA binding, especially through enrichment in RNA granules leading to high concentrations. Alternative models have been proposed, that place the RING domain closer to the center of the CC, allowing for RING dimerization within the TRIM25 dimer (47, 59) We note that our proposed mechanism of TRIM25 activation resembles the mechanism of RNA dependent regulation of the E3-ubiquitin ligase activity of roquins (60): Roquins feature two rigid multidomain motifs, that are connected by a flexible linker and both bind RNA. RNA binding therefore removes flexibility from the system and forces the protein into an active conformation. It should be noted, that in this case the effect is E2 dependent, so that RNA binding not only changes the extent of ubiquitination, but also specificity for certain chain compositions. Similar effects might also occur for TRIM25, as the close proximity of the RNA binding site and the RING domain in the above model combined with the necessity to accommodate both RNA and the E2~ubiquitin conjugate likely cause steric restrictions for the full complex. Such an interference of the RING domain with RNA binding could explain the earlier observation that RNA binding of full-length TRIM25 is weaker than that of CC-SPRY alone (26) .
In vitro liquid-liquid phase separation of TRIM25 upon RNA binding possibly explains TRIM25's previously observed subcellular localisation in RNA-granules upon overexpression or viral stress (7, 26) . Liquid-liquid phase-separated macromolecular condensates allow for free diffusion of moderately large molecules, including proteins, and exchange with the surrounding liquid while at the same time featuring very high protein concentrations and frequent encounters between proteins (61) .
In contrast to other previously observed mechanisms of oligomerisation in TRIM proteins, the RNA-induced liquid-liquid phase separation of TRIM25 does not require the B-Boxes, but the presence of the CC and PRY/SPRY domains is sufficient. This suggests possible mechanisms in which either more than one TRIM25 dimer can bind to one RNA molecule leading to a linear polymer or the PRY/SPRY domains of one TRIM25 molecule binds to the CC of a different molecule, an interaction that is strongly enhanced by RNA binding and could ultimately lead to formation of a cross-linked gel ( Figure 6B ). The necessity for binding of more than one TRIM25 dimer might explain why DENV-SL, but not the smaller pre-let-7 or lnczc3h7a stem loops induce phase separation. In this case, it should be expected that in the cellular context with RNA targets much longer than the minimal constructs used in this study, formation of TRIM25-RNA macromolecular condensates should be a common occurrence. Sanchez et al. noted that phase-separated condensates might form the necessary environment allowing for efficient interaction between TRIM25 and RIG-I and thereby resolve the apparent paradox that the TRIM25/RIG-I interaction is well established in vivo, but could not be reconstituted in vitro (26) .
Indeed, we found no evidence for a direct interaction between the TRIM25 PRY/SPRY and RIG-I CARDs in our NMR experiments, although these two domains alone are sufficient for coimmunoprecipitation from cells (15) . We noted however, that several of the mutants described to reduce the interaction between TRIM25 and RIG-I (F592, I594, L604 in murine TRIM25, corresponding to F597, I589 and L599 in human TRIM25) are located on or close to the second RNA binding site on the PRY/SPRY domain (42) . This suggests that RNA binding of both proteins to double-stranded segments in the same RNA could play a crucial role in RIG-I activation (Figure 7 ). This finding is supported by the recent identification of long non-coding RNAs that bind to both TRIM25 and RIG-I and facilitate their interaction (27, 44) . One of these RNAs is lnczc3h7a, that was described to interact with the RIG-I helicase domain independently of viral RNA and does not release autoinhibition of RIG-I (27) . However, this cannot explain how isolated RIG-I CARDs in vitro enrich in TRIM25/DENV-SL droplets, which requires either so far unreported RNA binding of the CARDs, an unreported interaction between CARDs and TRIM25 CC rather than PRY/SPRY or an allosteric change in TRIM25 upon RNA binding leading to increased affinity to CARDs.
Considering these new findings, we propose a mechanism, in which RNA-binding of TRIM25 not only assists in the recruitment of RIG-I through binding to the same RNA molecule or localization to phaseseparated condensates, but also directly activates the E3-ubiquitin ligase activity of TRIM25 by facilitating the interaction of the PRY/SPRY and CC domains (Figure 7) . Like RIG-I, many other substrates of TRIM25, e.g. ZAP, TUT4, MDM2 and p53 (23, 24, 49, 62, 63) , are putative RNA binding proteins and a mechanism that involves recruitment of these substrates through binding to the same RNA molecule or enrichment in RNA granules and activation of the TRIM25 E3 ligase activity through RNA-binding might be more universal.
Several E3 ligases other than TRIM25 promote ubiquitination of RIG-I leading to controversial discussions about their relative importance (64) (65) (66) (67) (68) (69) . Among these are several TRIM proteins (TRIM4, 15, 40) and Riplet, a close relative of TRIM25, that lost the B-Box domains and parts of the CC (70) (71) (72) . Mechanistically, a sequential ubiquitination of RIG-I by first Riplet in the CTD and then TRIM25 at the CARDs has been proposed (68) . It is noteworthy that although Riplet is closely related to TRIM25, it has lost the regions on the CC and in the L2 linker, which harbors RNA binding in TRIM25, and the RNA binding lysines and histidine found in the PRY/SPRY are not conserved. Interestingly, while the depends on the presence of both domains (2, 28) . This suggests that binding to the same RNA could also stabilize the TRIM25/NS1 interaction, thus deactivating TRIM25-mediated ubiquitination. A similar mechanism was recently proposed for the interaction of NS1 with DHX30 (74) . The inhibition of TRIM25 by other viral proteins such as paramyxovirus protein V and coronavirus protein N even depends only on their C-terminal domains that are also RBDs (4, 7, 75, 76) . RNA binding might therefore be widely exploited by viruses to inhibit TRIM25 and its crucial function in innate immunity. quantified and show a twenty-fold enrichment of RFP-CARDs in the droplets, while RFP enrichment is less than 2-fold. The T55I mutant reported to reduce the interaction of TRIM25 and RIG-I CARDs (77) has no significant impact on enrichment in the granules. 