PNPase knockout results in mtDNA loss and an altered metabolic gene expression program.

Eriko Shimada,Michael A Teitell,Carla M Koehler,Fasih M Ahsan,Brian D Gregory,Xiang Yu,Dana Case,Tara Teslaa,Sean Atamdede,Dian Huang,Mahta Nili,Benjamin J Perrin,Maria Moran

doi:10.1371/journal.pone.0200925

Abstract

Polynucleotide phosphorylase (PNPase) is an essential mitochondria-localized exoribonuclease implicated in multiple biological processes and human disorders. To reveal role(s) for PNPase in mitochondria, we established PNPase knockout (PKO) systems by first shifting culture conditions to enable cell growth with defective respiration. Interestingly, PKO established in mouse embryonic fibroblasts (MEFs) resulted in the loss of mitochondrial DNA (mtDNA). The transcriptional profile of PKO cells was similar to rho0 mtDNA deleted cells, with perturbations in cholesterol (FDR = 6.35 x 10−13), lipid (FDR = 3.21 x 10−11), and secondary alcohol (FDR = 1.04x10-12) metabolic pathway gene expression compared to wild type parental (TM6) MEFs. Transcriptome analysis indicates processes related to axonogenesis (FDR = 4.49 x 10−3), axon development (FDR = 4.74 x 10−3), and axonal guidance (FDR = 4.74 x 10−3) were overrepresented in PKO cells, consistent with previous studies detailing causative PNPase mutations in delayed myelination, hearing loss, encephalomyopathy, and chorioretinal defects in humans. Overrepresentation analysis revealed alterations in metabolic pathways in both PKO and rho0 cells. Therefore, we assessed the correlation of genes implicated in cell cycle progression and total metabolism and observed a strong positive correlation between PKO cells and rho0 MEFs compared to TM6 MEFs. We quantified the normalized biomass accumulation rate of PKO clones at 1.7% (SD ± 2.0%) and 2.4% (SD ± 1.6%) per hour, which was lower than TM6 cells at 3.3% (SD ± 3.5%) per hour. Furthermore, PKO in mouse inner ear hair cells caused progressive hearing loss that parallels human familial hearing loss previously linked to mutations in PNPase. Combined, our study reports that knockout of a mitochondrial nuclease results in mtDNA loss and suggests that mtDNA maintenance could provide a unifying connection for the large number of biological activities reported for PNPase.

Highlights

Polynucleotide phosphorylase (PNPase) is a conserved 3’-5’ exoribonuclease that bacteria and most eukarya express, but is absent in archae [1, 2]
We report that loss of PNPase, an RNA degrading enzyme that localizes in mitochondria, results in unanticipated depletion of the mitochondrial genome
Proliferation, and cell cycle features are similar between PNPase knockout (PKO) and rho0 mouse embryonic fibroblast (MEF), suggesting a role for PNPase in maintaining mitochondrial DNA (mtDNA)

Summary

Introduction

Polynucleotide phosphorylase (PNPase) is a conserved 3’-5’ exoribonuclease that bacteria and most eukarya express, but is absent in archae [1, 2]. In addition to phosphorolytic RNA degrading activity, bacterial PNPase catalyzes template independent polymerization of RNA [3, 4]. Similar to its bacterial counterpart, mammalian PNPase has several roles in RNA homeostasis, and it has been found to function within mitochondria. Mammalian PNPase exhibits enzymatic features that are similar to bacterial PNPase with a different optimal phosphate concentration for RNA degradation [28, 29]. In addition to RNASET2, which is a mitochondrial RNA degrading enzyme [35], recent studies have established that PNPase and the hSUV3 RNA helicase form a mtRNA degrading complex and degrade mirror-mtRNAs, which are noncoding mtRNAs that are antisense and complementary to coding mtRNAs [31, 34, 36]

Methods

Results

Conclusion