Highlights d RIPLET, not TRIM25, is the obligatory ubiquitin E3 ligase for RIG-I d RIPLET recognizes pre-assembled RIG-I oligomers on dsRNA and ubiquitinates RIG-I d RIPLET can cross-bridge RIG-I filaments formed on longer dsRNA d The two binding modes synergize for length dependent dsRNA discrimination by RIG-I

The mitochondrial contact site and cristae organizing system (MICOS) is crucial for the formation of crista junctions and mitochondrial inner membrane architecture. MICOS contains two core components. Mic10 shows membrane-bending activity, whereas Mic60 (mitofilin) forms contact sites between inner and outer membranes. Here we report that Mic60 deforms liposomes into thin membrane tubules and thus displays membrane-shaping activity. We identify a membrane-binding site in the soluble intermembrane space-exposed part of Mic60. This membrane-binding site is formed by a predicted amphipathic helix between the conserved coiled-coil and mitofilin domains. The mitofilin domain negatively regulates the membrane-shaping activity of Mic60. Binding of Mic19 to the mitofilin domain modulates this activity. Membrane binding and shaping by the conserved Mic60–Mic19 complex is crucial for crista junction formation, mitochondrial membrane architecture and efficient respiratory activity. Mic60 thus plays a dual role by shaping inner membrane crista junctions and forming contact sites with the outer membrane.

The conventional view posits that E3 ligases function primarily through conjugating ubiquitin (Ub) to their substrate molecules. We report here that RIPLET, an essential E3 ligase in antiviral immunity, promotes the antiviral signaling activity of the viral RNA receptor RIG-I through both Ub-dependent and -independent manners. RIPLET uses its dimeric structure and a bivalent binding mode to preferentially recognize and ubiquitinate RIG-I pre-oligomerized on dsRNA. In addition, RIPLET can cross-bridge RIG-I filaments on longer dsRNAs, inducing aggregate-like RIG-I assemblies. The consequent receptor clustering synergizes with the Ub-dependent mechanism to amplify RIG-I-mediated antiviral signaling in an RNA-length dependent manner.These observations show the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination. It also highlights a previously unrecognized mechanism by which the innate immune system measures foreign nucleic acid length, a common criterion for self vs. non-self nucleic acid discrimination. local concentration of 2CARD (Peisley et al., 2013). Second, in addition to RIG-I filament formation, K63-linked polyubiquitin (K63-Ub n ) was also shown to promote RIG-I signaling (Jiang et al., 2012). Structural studies further showed that K63-Ub n chains bind the periphery of the core 2CARD tetramer, bridging the adjacent subunits and stabilizing its assembly . The 2CARD tetramer then acts as a helical template to nucleate the MAVS filament formation for downstream signal activation (Wu and Hur, 2015).Despite the detailed understanding of the action of K63-Ub n on RIG-I, much remains debated as to how and when Ub is placed on RIG-I, which E3 ligase is involved and how it interplays with RIG-I filament formation. Previous studies reported that TRIM25 and RIPLET (i.e. RNF135) are two essential E3 ligases important for RIG-I signaling (Gack et al., 2007;Gao et al., 2009;Oshiumi et al., 2009;Oshiumi et al., 2010). A more recent study proposed that RIPLET and TRIM25 sequentially act on RIG-I upon viral RNA engagement . That is, RIPLET first acts on the C-terminal portion of RIG-I, releasing 2CARD, which is then modified by TRIM25. However, given the previous finding that RNA binding is sufficient to release 2CARD (Kowalinski et al., 2011), it was unclear whether RIPLET indeed acts to release 2CARD and if so, how. At the same time, accumulating evidence suggested that TRIM25 has multiple, RIG-I-independent antiviral functions (Choudhury et al., 2014;Li et al., 2017;Manokaran et al., 2015;Meyerson et al., 2017;Zheng et al., 2017), raising the question whether the observed effect of TRIM25 on RIG-I represents a direct or an indirect effect.We here report a combination of cellular and biochemical data showing that RIG-I activation is dependent on RIPLET, not TRIM25, and that RIPLET suffices to ubiquitinate and activate RIG-I. In addition, RIPLET recognizes the filamentous form of RIG-I using two distinct binding modes, the interplays of which o...