Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i⁶A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i⁶A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i⁶A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i⁶A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNA^Ser(UCN) mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i⁶A37 modification. These data demonstrate that deficiencies of i⁶A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs. Mitochondrial disorders are clinically diverse, and identifying the underlying genetic mutations is technically challenging due to the large number of mitochondrial proteins. Using high-throughput sequencing technology, we identified a disease-causing mutation in the TRIT1 gene. This gene encodes an enzyme, tRNA isopentenyltransferase, that adds an N^6-isopentenyl modification to adenosine-37 (i⁶A37) in a small number of tRNAs, enabling them to function correctly during the synthesis of essential mitochondrial proteins. We show that this mutation leads to severe deficiency of tRNA-i⁶A37 in the patient's cells that can be rescued by introduction of the wild-type TRIT1 protein. A deficiency in oxidative phosphorylation, the process by which energy (ATP) is generated in the mitochondria, leads to a mitochondrial disease presentation. Introducing the mutant protein into model yeast species and measuring the resulting impairment provided further evidence of the pathogenic effect of the mutation. Additional studies investigating a previously reported pathogenic mutation in a mitochondrial tRNA gene demonstrated that a mutation in a substrate of TRIT1 can also cause a loss of the modification, providing evidence of a new mechanism causing mitochondrial disease in humans.

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.

Using a combination of bioinformatic and molecular biology approaches a Drosophila melanogaster protein, DmTTF, has been identified, which exhibits sequence and structural similarity with two mitochondrial transcription termination factors, mTERF (human) and mtDBP (sea urchin). Import/processing assays indicate that DmTTF is synthesised as a precursor of 410 amino acids and is imported into mitochondria, giving rise to a mature product of 366 residues. Band-shift and DNase I protection experiments show that DmTTF binds two homologous, short, non-coding sequences of Drosophila mitochondrial DNA, located at the 3' end of blocks of genes transcribed on opposite strands. The location of the target sequences coincides with that of two of the putative transcription termination sites previously hypothesised. These results indicate that DmTTF is the termination factor of mitochondrial transcription in Drosophila. The existence of two DmTTF binding sites might serve not only to stop transcription but also to control the overlapping of a large number of transcripts generated by the peculiar transcription mechanism operating in this organism.

In mammals, NRF-2 (nuclear respiratory factor 2), also named GA-binding protein, is an Ets family transcription factor that controls many genes involved in cell cycle progression and protein synthesis as well as in mitochondrial biogenesis. In this paper, we analyzed the role of NRF-2 in the regulation of human genes involved in mitochondrial DNA transcription and replication. By a combination of bioinformatic and biochemical approaches, we found that the factor binds in vitro and in vivo to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-gamma, the DNA helicase TWINKLE, and the single-stranded DNA-binding protein mtSSB. The role of NRF-2 in modulating the expression of those genes was further established by RNA interference and overexpression strategies. On the contrary, we found that NRF-2 does not control the genes for the subunit A of DNA polymerase-gamma and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF proteins. Since NRFs are known to positively control the expression of transcription-activating proteins, the novelty emerging from our data is that proteins playing antithetical roles in mitochondrial DNA transcription, namely activators and repressors, are under different regulatory pathways. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GA-binding protein when searching for NRF-2 binding sites in the promoter of mitochondrial proteins.