Structural insights into the disease-linked human PNPase mutants in RNA binding and degradation
| Autor: | Golzarroshan, Bagher, 郭巴耳 |
|---|---|
| Rok vydání: | 2018 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 106 Polynucleotide phosphorylase (PNPase) is an evolutionary conserved 3'-to-5' exoribonuclease that functions in RNA processing and turnover in prokaryotes and eukaryotes. Human PNPase is mainly located in mitochondria where it not only participates in RNA processing and decay but it is also involved in importing a subgroup of structured RNAs, including 5S rRNA, MRP RNA, RNase P RNA and miRNAs, into the mitochondrial matrix. Mutations in PNPase that impair either mitochondrial RNA (mtRNA) degradation or RNA import are thus connected to mitochondrial dysfunctions and a wide spectrum of human diseases. Crystal structural studies reveal that PNPase is a trimeric protein assembled into a ring-like structure with a central channel for binding of a single-stranded RNA (ssRNA) and guiding its 3' end into the active site for degradation. However, it remained unclear how the disease-linked PNPase mutations affect protein folding, assembly and/or enzymatic activity. In this study, we show that the trimeric assembly of PNPase is disrupted by the disease-linked mutations, including Q378R and E475G. PNPase is oligomerized into a dimeric conformation after introducing the disease-linked mutations, and these PNPase mutants exhibit lower RNA-binding and degrading activities as compared to the wild-type protein. Moreover, we found that S1 domain of PNPase is responsible for the interaction with the stem-loop motif of imported RNAs but it is not involved in binding to ssRNA. We further determined the crystal structure of the dimeric form of the S1 domain-truncated PNPase with an N-terminal His-tag at a resolution of 2.8 Å showing that the KH domains are less accessible in the dimeric form of PNPase. The overall structures of the full-length trimeric and dimeric forms of PNPase were further determined by small-angle X-ray scattering (SAXS), showing that S1 domains are flexible with open to closed conformations in both conformers and that S1 domains are not fully accessible in the dimeric structure. Taken together these results explain why these dimeric PNPase mutants with less accessible RNA-binding KH and S1 domains interact with RNA poorly. We thus conclude that mutations at the interface of the trimeric PNPase tend to produce a dimeric protein with obstructed RNA-binding surfaces, thus impairing both of its RNA import and degradation activities and leading to mitochondria dysfunction and diseases. This study provides a possible strategy for the treatment of PNPase-associated diseases by stabilizing the trimeric conformation of PNPase that could improve its functions in both RNA import and decay in mitochondria. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|