A Mutation in PNPT1, Encoding Mitochondrial-RNA-Import Protein PNPase, Causes Hereditary Hearing Loss

Abstract

A subset of nuclear-encoded RNAs has to be imported into mitochondria for the proper replication and transcription of the mitochondrial genome and, hence, for proper mitochondrial function. Polynucleotide phosphorylase (PNPase or PNPT1) is one of the very few components known to be involved in this poorly characterized process in mammals. At the organismal level, however, the effect of PNPase dysfunction and impaired mitochondrial RNA import are unknown. By positional cloning, we identified a homozygous PNPT1 missense mutation (c.1424A>G predicting the protein substitution p.Glu475Gly) of a highly conserved PNPase residue within the second RNase-PH domain in a family affected by autosomal-recessive nonsyndromic hearing impairment. In vitro analyses in bacteria, yeast, and mammalian cells showed that the identified mutation results in a hypofunctional protein leading to disturbed PNPase trimerization and impaired mitochondrial RNA import. Immunohistochemistry revealed strong PNPase staining in the murine cochlea, including the sensory hair cells and the auditory ganglion neurons. In summary, we show that a component of the mitochondrial RNA-import machinery is specifically required for auditory function.

Functional Genomic Analysis of Human Mitochondrial RNA Processing

“…A mutation in FASTKD2 has been implicated in cytochrome c oxidase (COX) deficiency (Ghezzi et al, 2008), and we now find a 2-fold decrease of COX2-3,CYTB, ATP6/8, and ND3-4/4L-5 mt-mRNA expression under FASTKD2 silencing by at least one hairpin (Figure 2). PNPT1, which is implicated in both RNA degradation (Borowski et al, 2013) and 5S rRNA , RNase P RNA and MRP RNA import (Wang et al, 2010a), has also been recently associated with hearing loss and respiratory chain deficiency (Vedrenne et al, 2012; von Ameln et al, 2012). Our data reveals moderate increases in short-lived transcripts CYTB and ND2 as well as moderate decreases in long-lived transcripts COX1-3 (Figure 2) in PNPT1 silenced cells, consistent with previous reports and highlighting the complexity of this protein's role in the cell (Borowski et al, 2013).…”

Oocyte Factors Suppress Mitochondrial Polynucleotide Phosphorylase to Remodel the Metabolome and Enhance Reprogramming

“…Thus, the Tcl1-PnPase mechanism serves as a metabolic switch that is capable of remodeling the metabolome during reprogramming. Given this important role in regulating mitochondrial homeostasis, it is unsurprising that PnPase is required for mouse embryogenesis, muscle, brain, and inner ear development (Wang et al, 2010a;Vedrenne et al, 2012;von Ameln et al, 2012). Since mitochondrial numbers and functions are also tightly regulated in pre-fertilization oocytes and early embryonic development (Shyh-Chang et al, 2013b), the Tcl1-PnPase switch might be relevant not only for regulating mitochondria in iPSC reprogramming but also for mitochondrial replacement in aged oocytes and during in vitro fertilization.…”

EF-hand protein Ca 2+ buffers regulate Ca 2+ influx and exocytosis in sensory hair cells

“…Apical coils of the organs of Corti were stained for the mitochondria by using the marker PNPase (29) and ribbons (CtBP2; Fig. 7 A and B).…”

“…Immunohistochemistry was performed as described previously (19). To analyze the abundance of mitochondria, we fixed organs of Corti in methanol at −20°C for 20 min and double-stained hair cells for PNPase (mitochondria marker) (29) and CtBP2 (19) (to identify ribbons). In all other cases, organs were fixed with 4% (wt/vol) formaldehyde for 10-60 min on ice.…”