ABSTRACT
Expression of human mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. The mitochondrial genome encodes 22 tRNAs, 2 rRNAs and 11 mRNAs and their post-transcriptional modification constitutes one of the key regulatory steps during mitochondrial gene expression. Cytosine-5 methylation (m5C) has been detected in mitochondrial transcriptome, however its biogenesis has not been investigated in details. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterized as an RNA methyltransferase introducing m5C in nuclear-encoded tRNAs, mRNAs and microRNAs and associated with cell proliferation and differentiation, with pathogenic variants in NSUN2 being linked to neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation-knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells-we show that NSUN2 is necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.
Subject(s)
5-Methylcytosine/metabolism , Eczema/genetics , Growth Disorders/genetics , Intellectual Disability/genetics , Methyltransferases/genetics , Microcephaly/genetics , RNA Processing, Post-Transcriptional , RNA, Mitochondrial/genetics , RNA, Transfer/genetics , Animals , CRISPR-Cas Systems , Eczema/metabolism , Eczema/pathology , Facies , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Editing , Gene Knockout Techniques , Growth Disorders/metabolism , Growth Disorders/pathology , HEK293 Cells , Humans , Intellectual Disability/metabolism , Intellectual Disability/pathology , Methylation , Methyltransferases/deficiency , Mice , Mice, Knockout , Microcephaly/metabolism , Microcephaly/pathology , Mitochondria/genetics , Mitochondria/metabolism , Nucleic Acid Conformation , Oxidative Phosphorylation , Primary Cell Culture , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Mitochondrial/metabolism , RNA, Transfer/metabolismABSTRACT
Mutations of the mitochondrial genome (mtDNA) underlie a substantial portion of mitochondrial disease burden. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation and prognosis. To address the lack of effective treatment for these disorders, we exploited a recently developed mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease in cardiac tissue: the m.5024C>T tRNAAla mouse. Through application of a programmable nuclease therapy approach, using systemically administered, mitochondrially targeted zinc-finger nucleases (mtZFN) delivered by adeno-associated virus, we induced specific elimination of mutant mtDNA across the heart, coupled to a reversion of molecular and biochemical phenotypes. These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin.
Subject(s)
Gene Editing , Mitochondria, Heart/genetics , Mitochondrial Diseases/genetics , Zinc Finger Nucleases/genetics , Animals , DNA, Mitochondrial/genetics , Dependovirus/genetics , Disease Models, Animal , Humans , Mice , Mitochondria, Heart/pathology , Mitochondrial Diseases/pathology , Mitochondrial Diseases/therapy , Mutation/genetics , Prognosis , RNA, Transfer/genetics , Zinc Finger Nucleases/therapeutic useABSTRACT
Mitochondrial diseases often result from mutations in the mitochondrial genome (mtDNA). In most cases, mutant mtDNA coexists with wild-type mtDNA, resulting in heteroplasmy. One potential future approach to treat heteroplasmic mtDNA diseases is the specific elimination of pathogenic mtDNA mutations, lowering the level of mutant mtDNA below pathogenic thresholds. Mitochondrially targeted zinc-finger nucleases (mtZFNs) have been demonstrated to specifically target and introduce double-strand breaks in mutant mtDNA, facilitating substantial shifts in heteroplasmy. One application of mtZFN technology, in the context of heteroplasmic mtDNA disease, is delivery into the heteroplasmic oocyte or early embryo to eliminate mutant mtDNA, preventing transmission of mitochondrial diseases through the germline. Here we describe a protocol for efficient production of mtZFN mRNA in vitro, and delivery of these into 0.5 dpc mouse embryos to elicit shifts of mtDNA heteroplasmy.
