RNASEH1 Mutations Impair mtDNA Replication and Cause Adult-Onset Mitochondrial Encephalomyopathy

Abstract

Chronic progressive external ophthalmoplegia (CPEO) is common in mitochondrial disorders and is frequently associated with multiple mtDNA deletions. The onset is typically in adulthood, and affected subjects can also present with general muscle weakness. The underlying genetic defects comprise autosomal-dominant or recessive mutations in several nuclear genes, most of which play a role in mtDNA replication. Next-generation sequencing led to the identification of compound-heterozygous RNASEH1 mutations in two singleton subjects and a homozygous mutation in four siblings. RNASEH1, encoding ribonuclease H1 (RNase H1), is an endonuclease that is present in both the nucleus and mitochondria and digests the RNA component of RNA-DNA hybrids. Unlike mitochondria, the nucleus harbors a second ribonuclease (RNase H2). All affected individuals first presented with CPEO and exercise intolerance in their twenties, and these were followed by muscle weakness, dysphagia, and spino-cerebellar signs with impaired gait coordination, dysmetria, and dysarthria. Ragged-red and cytochrome c oxidase (COX)-negative fibers, together with impaired activity of various mitochondrial respiratory chain complexes, were observed in muscle biopsies of affected subjects. Western blot analysis showed the virtual absence of RNase H1 in total lysate from mutant fibroblasts. By an in vitro assay, we demonstrated that altered RNase H1 has a reduced capability to remove the RNA from RNA-DNA hybrids, confirming their pathogenic role. Given that an increasing amount of evidence indicates the presence of RNA primers during mtDNA replication, this result might also explain the accumulation of mtDNA deletions and underscores the importance of RNase H1 for mtDNA maintenance.

Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria

“…6A), as reported previously (14). The earlier report also measured a two-to fourfold increase in mitochondrial replication intermediates in the mutant fibroblasts detected by 2D gel-electrophoresis.…”

“…The V142I substitution results in lower RNase H activity than the wild-type enzyme (14) but is associated with a marked decrease in R-loops in the fibroblasts of the patient studied here (Fig. 6C and SI Appendix, Fig.…”

“…Inactivation by oxidation would be desirable if the V142I substitution disrupted RNase H1's interaction with a protein partner(s) that restricted its actions. Even at 40% of full activity (14), unregulated V142I RNase H1 would be expected to degrade RNA/DNA hybrids inappropriately and thereby could increase the rate of turnover of mitochondrial R-loops. All that said, a definitive demonstration that R-loop depletion is the result of V142I RNase H1 awaits the creation of a cellular or animal model with this mutation.…”

“…Concordant with the mtDNA copy number data, the RNase H1 variant V142I produces milder pathology in humans than seen with gene ablation in the mouse: adult-onset neuromuscular disease (encephalomyopathy) (this report and ref. 14), as opposed to embryonic lethality (13). However, analysis of the steady-state level of the protein using detergent-solubilized cell lysates, stored frozen, implied that the V142I substitution resulted in almost complete loss of RNase H1, because the protein was barely detectable (Fig.…”

Human mitochondrial DNA replication machinery and disease

“…A recent study by Reyes et al examined three families with recessive inheritance patterns consistent with affected individuals harboring causative homozygous or compound-heterozygous mutations [78]. Whole-exome sequencing revealed mutations in the RNASEH1 gene.…”