Abstract
Human polynucleotide phosphorylase (hPNPase) is a 3'-to-5' exoribonuclease located in mitochondria, where it plays crucial roles in RNA degradation and RNA import. Mutations in hPNPase can impair these functions, leading to various mitochondrial dysfunctions and diseases. However, the mechanisms by which hPNPase switches between its roles as an RNA-degrading enzyme and an RNA carrier, as well as how disease-associated mutations may affect these distinct functions, remain unclear. In this study, we present cryo-electron microscopy structures of hPNPase, highlighting the flexibility of its S1 domains, which cap the ring-like RNA-degradation chamber and shift between two distinctive open and closed conformations. We further demonstrate by small-angle X-ray scattering and biochemical analyses that the disease-associated mutations P467S and G499R impair hPNPase's stem-loop RNA-binding and degradation activities by limiting the S1 domain's ability to transition from an open to closed state. Conversely, the D713Y mutation, located within the S1 domain, does not affect the RNA-binding affinity of hPNPase, but diminishes its interaction with Suv3 helicase for cooperative degradation of structured RNA. Collectively, these findings underscore the critical role of S1 domain mobility in capturing structured RNA for degradation and import, as well as its involvement in mitochondrial degradosome assembly. Our study thereby reveals the molecular mechanism of hPNPase in RNA binding and degradation, and the multiple molecular defects that could be induced by disease-linked mutations in hPNPase.