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

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.

Import of Non-Coding RNAs into Human Mitochondria: A Critical Review and Emerging Approaches

“…In IMS, it is believed to be a factor in RNA import in the mitochondria [19], to control the escape of mitochondrial double-stranded RNA into the cytosol under physiological and disease conditions [108], and may, upon release into the cytosol, also play a role in apoptosis [109]. Mutations in human PNPase are linked to mitochondrial diseases of varying severities, characterized by respiratory chain deficiencies and associated with both mitochondrial RNA processing defects and perturbed RNA import [110,111,112,113]. Given the pervasive impact of PNPase on the mitochondrial RNA metabolism, it is not always possible to disentangle the observed phenotypes and conclusively connect them with various functions proposed for this protein.…”

“…However, the pore is too narrow to allow the passage of a structured RNA. A more recent study has proposed that the S1 domains, also forming a pore atop of the KH domains, interact with structured double-stranded RNAs [113]. If the length of the 3′ overhang of the bound RNA is too short, it will prevent the entry of the RNA into the KH pore and the PH channel, protecting it from degradation (Figure 4b) [19,115].…”

“…(Left) If the 3′ overhang is long enough, the bound RNA can access the active site (AS) and be degraded. (Right) Structured transcripts, e.g., imported small noncoding RNAs cannot enter the KH pore, which prevents their degradation [113,115].…”

Clinical Spectrum and Functional Consequences Associated with Bi-Allelic Pathogenic PNPT1 Variants

“…PNPase has been suggested to play a role in RNA import into mitochondria [3,4]; however, experimental data have been contradictory and, to date, there is no general agreement about an RNA import mechanism [5]. Recent reports suggest that disrupted PNPase RNA processing could lead to the accumulation of double-stranded mtRNAs, with the possibility of triggering an altered immune response [6,7].…”

Structural insights into nanoRNA degradation by human Rexo2

“…A number of human ribonucleases and helicases participate in mitochondrial RNA (mtRNA) decay, including polynucleotide phosphorylase (PNPase), Suv3 helicase and RNA exoribonuclease 2 (Rexo2, also named small fragment nuclease, SFN). PNPase forms a complex with the helicase Suv3 to cooperatively degrade mtRNA with secondary structures (Minczuk et al 2002;Wang et al 2009), producing final cleavage products of ∼4 nucleotides (nt) (Lin et al 2012;Golzarroshan et al 2018). In contrast, Rexo2 likely acts as a scavenger, degrading small single-stranded RNA of <5 nt (referring to as "nanoRNAs") and generating monoribonucleotides for RNA salvage in mitochondria (Goldman et al 2011;Bruni et al 2013).…”