RNA helicases and E3 ubiquitin ligases mediate many critical functions in cells, but their actions have largely been studied in distinct biological contexts. Here, we uncover evolutionarily conserved rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryoelectron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement may apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses suggest not only conserved substrates for TRIM proteins but also, unexpectedly, deep evolutionary connections between TRIM proteins and RNA helicases, linking ubiquitin and RNA biology throughout animal evolution. Display omitted •TRIM65 selectively recognizes MDA5 filaments using PSpry bivalency•TRIM65 PSpry binds α1/α3 helices in the Hel2 domain of MDA5•RIPLET recognizes RIG-I using a similar epitope in the helicase domain•Distinct TRIM proteins recognize common epitopes in diverse helicases using bivalency Proper immune function requires multiple layers of checks and balances. Kato et al. show a conserved mechanism by which the antiviral proteins RIG-I-like receptors collaborate with a family of E3 ligases, TRIM-like proteins, to ensure high fidelity and robustness of the antiviral immune response.