ZNFX1, an immunoregulatory RNA helicase and E3 ubiquitin ligase, assembles into pleiomorphic polymers.

NFX1-type zinc-finger-containing 1 (ZNFX1) is an SF1-family RNA helicase essential for innate immunity. Patients with ZNFX1 mutations experience recurrent infections, yet the underlying mechanism remains unclear. We determined cryo-electron microscopy (cryo-EM) structures of RNA-bound and RNA-free ZNFX1 and revealed auto-inhibition of the helicase-associated ATPase through a regulatory insertion occluding the RNA-binding groove. ZNFX1 also functions as a bi-catalytic E3 ubiquitin ligase, containing an RZ finger homologous to RNF213 and a previously unidentified Miz-like domain, which cooperate in catalyzing ubiquitylation. Ubiquitin chains enhance E3 activity further. Patient mutations demonstrate that the helicase/ATPase, E3 ligase, and the rigid zinc-finger stalk connecting them are required for function. ZNFX1 can assemble into structured, pleiomorphic polymers fostering trans-auto-ubiquitylation; understanding this reveals a mechanism that controls ZNFX1 stability and may facilitate RNA sequestration in stress granules and antiviral activity. These findings establish ZNFX1 as a multifunctional enzyme in immunity that couples RNA sensing to ubiquitin signaling and assembles into higher-order structures for signal amplification.