An Upstream Open Reading Frame and the Context of the Two AUG Codons Affect the Abundance of Mitochondrial and Nuclear RNase H1

Suzuki, Yutaka; Holmes, J. Bradley; Cerritelli, Susana M.; Sakhuja, Kiran; Minczuk, Michal; Holt, Ian; Crouch, Robert J.

doi:10.1128/mcb.00619-10

Cited by 86 publications

(88 citation statements)

References 37 publications

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“…The fragments were GFP-senataxin , GFP-senataxin , GFPsenataxin 624 -2146 , and GFP-senataxin 2099 -2677 . The GFP-tagged RNase H1 expression vector GFP-M27-H1 is described elsewhere (48,49). HeLa cells were grown on coverslips in a 10-cm plate and transfected for 24 h with 6 g DNA using Lipofectamine 2000 (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Senataxin, Defective in the Neurodegenerative Disorder Ataxia with Oculomotor Apraxia 2, Lies at the Interface of Transcription and the DNA Damage Response

Yüce

West

2013

Molecular and Cellular Biology

176

161

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The neurodegenerative disorder ataxia with oculomotor apraxia 2 (AOA-2) is caused by defects in senataxin, a putative RNA/ DNA helicase thought to be involved in the termination of transcription at RNA polymerase pause sites. RNA/DNA hybrids (R loops) that arise during transcription pausing lead to genome instability unless they are resolved efficiently. We found that senataxin forms distinct nuclear foci in S/G 2 -phase human cells and that the number of these foci increases in response to impaired DNA replication or DNA damage. Senataxin colocalizes with 53BP1, a key DNA damage response protein, and with other factors involved in DNA repair. Inhibition of transcription using ␣-amanitin, or the dissolution of R loops by transient expression of RNase H1, leads to the loss of senataxin foci. These results indicate that senataxin localizes to sites of collision between components of the replisome and the transcription apparatus and that it is targeted to R loops, where it plays an important role at the interface of transcription and the DNA damage response.

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Section: Methodsmentioning

confidence: 99%

Senataxin, Defective in the Neurodegenerative Disorder Ataxia with Oculomotor Apraxia 2, Lies at the Interface of Transcription and the DNA Damage Response

Yüce

West

2013

Molecular and Cellular Biology

176

161

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“…An upstream AUG, encoding Met0, competes with initiation at Met1 (which begins the mitochondrial variant), so that in physiological conditions, the nuclear form (which starts with Met27) is predominant. 7 An N-terminal MTS targets this protein to mitochondria and is eventually cleaved off by the mitochondrial matrix peptidase after translocation, resulting in a protein almost identical to the nuclear form. A second, exclusively nuclear ribonucleotidase affecting RNA-DNA hybrids is RNase H2, alterations in which are responsible for Aicardi-Goutieres syndrome (MIM: 610333, 610181).…”

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confidence: 99%

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

Reyes

Melchionda

Nasca

et al. 2015

The American Journal of Human Genetics

107

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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.

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“…Mitochondrial isolation from HEK293 cells was performed as described 31 . In short, cells were homogenized on ice with a Potter-Elvehjem homogenizer in a buffer consisting of 2 mM KCl, 1.5 mM MgCl, 1 mg ml À 1 BSA and 20 mM HEPES, pH 8.0.…”

Section: Methodsmentioning

confidence: 99%

Human copper chaperone for superoxide dismutase 1 mediates its own oxidation-dependent import into mitochondria

et al. 2013

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Oxidative stress is counteracted by various cellular systems, including copper-zinc superoxide dismutase 1 (SOD1) and its activating chaperone, that is, the copper chaperone for SOD1 (CCS1). Both enzymes are structurally related, and both localize to the cytosol and the mitochondrial intermembrane space where they specifically counteract mitochondria-derived superoxide. The mechanism by which human CCS1 is transported into mitochondria is largely unclear. Here we show that CCS1 import depends on the presence of mature CCS1 in the mitochondria. During import, a disulphide bond is formed in CCS1 in a CCS1-dependent reaction. We demonstrate that oxidation and import depend on the presence of cysteine residues at positions 227 and 141/144 in CCS1. Notably, CCS1 import parallels SOD1 import that also depends on CCS1. Our observations suggest that CCS1 serves as a specialized import receptor in mitochondria that facilitates the import and folding of SOD1 and CCS1, thereby extending the substrate spectrum of oxidation-dependent protein import in the mitochondrial intermembrane space.

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An Upstream Open Reading Frame and the Context of the Two AUG Codons Affect the Abundance of Mitochondrial and Nuclear RNase H1

Cited by 86 publications

References 37 publications

Senataxin, Defective in the Neurodegenerative Disorder Ataxia with Oculomotor Apraxia 2, Lies at the Interface of Transcription and the DNA Damage Response

Senataxin, Defective in the Neurodegenerative Disorder Ataxia with Oculomotor Apraxia 2, Lies at the Interface of Transcription and the DNA Damage Response

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

Human copper chaperone for superoxide dismutase 1 mediates its own oxidation-dependent import into mitochondria

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