Cryo-EM Structure of Substrate-Engaged Nuclear Exosome Targeting (Next) Complex

Abstract

The nuclear exosome targeting (NEXT) complex recruits a diverse set of coding and noncoding RNA for quality control and degradation by the ribonucleolytic RNA exosome. It consists of an RNA adaptor RBM7, a zinc knuckle scaffold protein ZCCHC8, and a superfamily 2 Ski2-like helicase MTR4. Genetic lesions in subunits of the NEXT complex have been associated with cancer and neurological diseases. To illuminate the molecular basis for RNA targeting of the NEXT complex, we reconstituted the human NEXT core complex and characterized its biochemical activities and structure. NEXT displays enhanced helicase activity compared to MTR4 alone in a strand displacement assay. Optimal unwinding activity was observed with substrates containing a short 3′ poly(A) tail and an upstream polyuridine sequence for interaction with RBM7. Cryo-EM structures of substrate-engaged NEXT reveal a dimeric assembly mediated by homotypic interactions between the N-terminal domains of ZCCHC8. ZCCHC8 interacts with the MTR4 KOW domain and tethers RBM7 to the RecA2 domain of MTR4. The single-stranded region of the substrate is proximal to RBM7 with the 3′ tail captured within the helicase core of MTR4. Ongoing analysis of disease-linked variants of NEXT subunits will be presented in light of mechanisms that may contribute to pathogenesis.