Abstract
PNPase (polynucleotide phosphorylase) is an evolutionarily conserved enzyme that participates in RNA processing and degradation in almost all species from bacteria to plants and higher mammals. Human PNPase (hPNPase) not only degrades specific mRNA and miRNA, but also imports RNA into mitochondria, and thus it regulates diverse physiological processes, including cellular senescence and homeostasis. It has been shown that hPNPase forms a complex with the helicase hSUV3 which promotes the activity of hPNPase in the digestion of structural RNA. However, how hPNPase binds and cleaves RNA using its various domains, as well as how hSUV3 interacts and works with hPNPase in RNA degradation are mostly unknown. Here we determined the crystal structures of an S1 domain-truncated hPNPase and hSUV3 at a resolution of 2.1 Å and 3.3 Å, respectively. hPNPase contains one α-helical, one KH, one S1, and two RNase PH domains. Based on previous studies, the KH and S1 domain are involved in RNA binding and RNase PH domain is the active center for RNA degradation. We showed here that the trimeric hPNPase has a hexameric ring-like structure formed by six RNase PH domains, capped with a trimeric KH pore. Our biochemical and mutagenesis studies suggest that the S1 domain is not critical for RNA binding, and conversely, that the conserved GXXG motif in the KH domain directly participates in RNA binding in hPNPase. Our studies thus provide structural and functional insights into hPNPase, which uses a KH pore to trap a long RNA 3' tail that is further delivered into an RNase PH channel for the degradation process. Structural RNA with short 3' tails are, on the other hand, transported but not digested by hPNPase. Moreover, we found that hPNPase forms a 3:3 and 3:2 complex with hSUV3. The crystal structure of hSUV3 reveals a four domain structure, the N-terminal domain, two RecA-like domains, and the C-terminal domain which is not only involved in nucleic acids binding, but also in interacting with hPNPase. The crystal structure of hSUV3 thus offers a starting point for the future study of its interactions with hPNPase. In summary, our studies provide structural insights for the role of hPNPase in RNA degradation and transportation.