Crystal structure of dimeric human PNPase reveals why disease-linked mutants suffer from low RNA import and degradation activities

• Published in: Nucleic acids research 2018-09, Vol.46 (16), p.8630-8640
• Main Authors: Golzarroshan, Bagher, Lin, Chia-Liang, Li, Chia-Lung, Yang, Wei-Zen, Chu, Lee-Ya, Agrawal, Sashank, Yuan, Hanna S
• Format: Article
• Language: English
• Subjects: Structural Biology
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gky642

Abstract Human polynucleotide phosphorylase (PNPase) is an evolutionarily conserved 3′-to-5′ exoribonuclease principally located in mitochondria where it is responsible for RNA turnover and import. Mutations in PNPase impair structured RNA transport into mitochondria, resulting in mitochondrial dysfunction and disease. PNPase is a trimeric protein with a doughnut-shaped structure hosting a central channel for single-stranded RNA binding and degradation. Here, we show that the disease-linked human PNPase mutants, Q387R and E475G, form dimers, not trimers, and have significantly lower RNA binding and degradation activities compared to wild-type trimeric PNPase. Moreover, S1 domain-truncated PNPase binds single-stranded RNA but not the stem-loop signature motif of imported structured RNA, suggesting that the S1 domain is responsible for binding structured RNAs. We further determined the crystal structure of dimeric PNPase at a resolution of 2.8 Å and, combined with small-angle X-ray scattering, show that the RNA-binding K homology and S1 domains are relatively inaccessible in the dimeric assembly. Taken together, these results show that mutations at the interface of the trimeric PNPase tend to produce a dimeric protein with destructive RNA-binding surfaces, thus impairing both of its RNA import and degradation activities and leading to mitochondria disorders.