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

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.

Polynucleotide phosphorylase: Not merely an RNase but a pivotal post-transcriptional regulator

“…Equally exciting were the discoveries that hPNPase mediates the importation of RNA into the mitochondrial matrix [ 25 ] and degrades mRNAs and polyadenylated noncoding RNAs upon release into the cytoplasm following mitochondrial outer membrane permeabilization during apoptosis [ 120 ]. Given the recent discovery that PNPase is critical for mitochondrial DNA maintenance [ 126 ], hPNPase is vital to the proper replication and function of mitochondria and to human life. Considering that these recent discoveries of additional functions for PNPase were made after more than a half century of study, we expect to see many more exciting findings reported on this ancient enzyme in the years to come.…”

“…Both RNases serve critical roles in mitochondria, in which RNase MRP cleaves the RNA primers used for mitochondrial DNA replication and RNase P processes the large mitochondrial polycistronic transcripts that give rise to 22 tRNAs, 12S and 16S rRNAs, as well as 13 mRNAs encoding electron transport chain (ETC) components involved in oxidative phosphorylation (reviewed in [ 125 ]), i.e., the synthesis of ATP that is powered by the transfer of electrons from NADH or FADH 2 to O 2 . Likewise, a stable PNPase knockout in mouse embryonic fibroblasts resulted in the loss of both mitochondrial DNA and cellular respiration, supporting a role for PNPase in mitochondrial DNA maintenance [ 126 ]. The vital function of hPNPase in facilitating proper expression of the ETC components is further evidenced by the fact that knockdown of PNPase in HEK293 cells leads to impairment of the ETC and disruption of oxidative phosphorylation [ 117 ].…”

The identification of mecciRNAs and their roles in mitochondrial entry of proteins

“…To exclude potential misidentification of junction sites due to differences in annotated genome and genome of cultured cells, nuclear DNA and mitochondrial DNA from HeLa cells were re-sequenced, and no DNA read matching junction sequences of human mecciRNAs was identified ( Figure S3A). In previously published RNA-seq data of cells without mitochondria (rho0 MEF cells), no mecciRNA was identified, whereas mecciRNAs were found in wildtype MEF cells (Shimada et al, 2018) ( Figure S3B). Interestingly, considering the small size of mitochondrial genome, mecciRNAs were relatively enriched in nascent circRNAs detected from HeLa cells (Bao et al, 2018) (Figure S3C).…”

“…PNPASE may be simply required for the mitochondrial importation and exportation of mecciRNAs, and thus to affect protein importation indirectly. PNPASE plays essential roles in mitochondria, and a recent study shows that knockout of PNPASE gene in cultured cells leads to depletion of mtDNA, and the cells eventually are absent of mitochondria (rho0 cells) (Shimada et al, 2018). The relevance of PNPASE to mecciRNAs and the mitochondrial protein importation extends the understandings about critical roles of PNPASE.…”

Mitochondrial Import of Malat1 Regulates Cardiac Mitochondrial Function in Type 2 Diabetes Mellitus through Interaction with MicroRNA-23b

“…In 2010, Wang et al rst established an RNA import function for PNPase in mammalian systems [24], revealing that tRNA and ribozyme machinery are transported by PNPase into the mitochondrion. Additionally, changes in PNPase expression have resulted in alterations in cellular function and miRNA import [6,25]. PNPase, through its KH and S1 RNA binding domains, may provide a functional context for ncRNA import and regulation in the mitochondrion.…”