None of the RNA-modulated MAVS interactors were known to regulate RLR signaling or IFN production. So we used a targeted siRNA screening approach to find out if they do! We identified 14 new regulators of RLR signaling that bind to MAVS in an RNA-modulated manner.

But how does RNA modulate MAVS function? We hypothesized that RNA could alter protein-protein interactions at the MAVS signalosome. So we performed IP-MS of MAVS -/+ RNase and identified several factors with decreased or increased MAVS interaction in the absence of RNA.

We then using APOBEC1-mediated profiling to identify the RNAs that MAVS interacts with. It binds primarily to the 3’ UTRs of ~100 mRNAs!! Plus, he have hints that the complement of MAVS-associated RNAs may be altered during infection!

This central region is predicted to be completely disordered - this is why we don't have crystal/cryoEM structures of this part of MAVS. What’s more, this disorder is highly conserved, even if specific residues are not. Disordered protein domains tend to interact with RNA!!

But what domains of MAVS are responsible for RNA interaction? Not the N-terminal CARD or the C-terminal TM helix (data not shown (read preprint)). However, deletion of the central region of MAVS between amino acids 103-467 abolishes MAVS-RNA interactions.

Using infrared-dye crosslinking and immunoprecipitation, we found that overexpressed and endogenous MAVS directly interacts with RNA! MAVS RNA-binding is conserved for murine MAVS.

We found that MAVS and other signalosome components have an RNase-dependent shift in their migration through a sucrose gradient. Higher-order MAVS complexes in dense fractions diminish with RNase treatment (look at fraction 10!). This suggests that the MAVS signalosome is associated with RNA.

But does RNA regulate MAVS function? We found that RNase can inhibit IRF3 phosphorylation by Sendai virus-induced MAVS signalosomes in vitro. This also holds true when the MAVS signalosome is activated by a non-RNA molecule in the absence of viral RNA. Cellular RNA promote MAVS signalosome activity!

Localization, oligomerization, protein-protein interactions, and PTMs all regulate the MAVS signalosome. But we asked, “could RNA play a role too?” This is because RNA is versatile and the best biomolecule there is. Without a doubt.

Higher-order multi-protein complexes centered on oligomerized adaptors are a feature of immune signaling. One of these complexes, the MAVS signalosome, forms at contact sites between mitochondria, peroxisomes, and ER when RLRs sense foreign RNA.

I hope you enjoy the BS-orial (uh... that doesn't work) below and read the preprint. But here’s the TLDR: