POLRMT mutations impair mitochondrial transcription causing neurological disease

Abstract

While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.

Fig. 1. Molecular genetics and histochemical studies.

a Family pedigrees and segregation status of POLRMT patients. Affected individuals are depicted in black, circles represent females, squares represent males; a diagonal line through the symbol represents deceased individuals and double horizontal lines indicate consanguinity. b Diagnostic histochemical analyses (n = 2) of skeletal muscle-derived from P3 showing (i) Haematoxylin & Eosin staining (ii) SDH (iii) COX and (iv) COX-SDH reactivities. The sequential COX-SDH reaction in (iv) revealed a mosaic pattern of COX-deficiency in P3. The scale bar shown is 100 um.

Fig. 2. Domain organisation of POLRMT.

a The structure of the transcription initiation complex is shown bound to the heavy strand promoter (HSP) (PDB ID: 6ERQ). POLRMT comprises four main domains: the N-terminal extension (yellow), a pentatricopeptide repeat (PPR) domain (purple), the N-terminal domain (green), and the C-terminal domain (orange). The positions of the disease-causing variants analysed in this study are indicated (black circles) as well as the location of the active site (full black circle). b, c Interactions formed between the residues implicated in disease (magenta, bold text) and surrounding residues (green). b P1 harbours the compound heterozygous variants D870N and P566S/S1193F, P2 harbours the H250D variant as well as a deletion of residues 742–747 and P3 harbours a deletion of residues 881–883. c P4 harbours the S611F variant, P5 harbours the F641L variant, P6 harbours the P810S variant, and P7/8 harbour the R1013C variant. Structures in (b, c) are generated from the transcription initiation complex (PDB ID: 6ERP) and transcription elongation complex (PDB ID: 5OLA).

Fig. 3. mtDNA, mitochondrial transcript, and OXPHOS protein levels in POLRMT patient fibroblasts.

a Effects of the patient variants on mtDNA levels in vivo were analysed by Southern blot (left panel) and qPCR (right panel). No significant deletions/depletion of mtDNA were observed in P1, P2, and P6 while mild mtDNA depletion was observed in P3 when compared to control (Ct). Southern blotting was repeated three times and a representative example is shown. For qPCR analysis, 6 technical repeats were performed (patient cells were grown in two separate cell cultures, which were each analysed three times). b Levels of mitochondrial transcripts in patient fibroblasts were analysed by RT-qPCR. P1, P2, and P6 showed a mild down-regulation of gene expression whereas P3 showed a severe loss of mitochondrial transcripts (5–32% of wild-type levels). For qPCR analysis, 4–6 technical repeats were performed (patient cells were grown in two separate cell cultures, which were each analysed 2–3 times). c Western blot analysis of whole-cell lysates extracted from adult control (Ct1, Ct2) and POLRMT patient (P1, P2, P3, P6) fibroblasts (left panel). P1 and P3 showed a decrease in the steady-state levels of subunits of CI (NDUFB8), CIII (UQCRC2), and CIV (COXII). Normal levels of OXPHOS subunits were detected in P2 and P6. One-dimensional BN PAGE analysis revealed a combined OXPHOS assembly defect in P3 and a slight defect in the assembly of CIV in P1 (right panel). The nuclear-encoded SDHA and/or SDHB subunits were used as loading controls. Representative blots of three independent SDS and BN PAGE experiments for P1, P2, and P3 are shown. One representative western blot experiment out of two independent analysis is shown for P6.

Fig. 4. In vitro transcription assays of wild-type and mutant POLRMT.

a Combination of mutant alleles present in individual patients. b Promoter-dependent transcription from a linear LSP template comparing the activities of individual mutant POLRMT variants. The 400 nt run-off product (LSP RO) is indicated along with the pre-terminated transcription products formed at CSB II. Each POLRMT variant was analysed at least 3 times and a representative experiment is shown. c Promoter-dependent transcription as in panel b, but now using the combination of mutations identified in affected patients. The levels of run-off products relative to wild-type are indicated above each lane. Each POLRMT combination was analysed at least 3 times and a representative experiment is shown. d Single ribonucleotide incorporation (SNRI) assay comparing the combination of mutations identified in affected patients (top panel). Percentage of RNA product relative to the labelled substrate is indicated in the bar graph below. Data are presented as mean ± SEM of three individual experiments. Unpaired t test was performed to check significance. Asterisks represent statistically significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.005). e Primer synthesis on template containing the OriL stem loop structure with the indicated POLRMT variants (top panel) revealed mild to strong impairment of primase activity compared to the wild-type (middle panel). In the bar graph (lower panel), the percentage activity relative to wild-type is shown for each lane. Data are presented as mean ± SEM of three individual experiments. The P value was calculated as described in panel (d).