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The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication

ABSTRACT: The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication

PROVIDER: PRJNA669032 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12819267.fastq.gz	Fastqsanger.gz
	SRR12819268.fastq.gz	Fastqsanger.gz
	SRR12819269.fastq.gz	Fastqsanger.gz
	SRR12819270.fastq.gz	Fastqsanger.gz
	SRR12819271.fastq.gz	Fastqsanger.gz

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The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication

Project description:Single-stranded DNA (ssDNA) binding proteins protect regions of ssDNA formed during processes such as DNA replication and repair. We here devise a genetic screen and identify the mitochondrial ssDNA-binding protein (mtSSB) as a key regulator of mtDNA levels. In mitochondria, RNA synthesis from the light-strand promoter (LSP) is required for transcription as well as for generating the primers for initiation of mtDNA synthesis. We find that mtSSB is essential for mtDNA replication initiation, as transcription is strongly upregulated from the LSP in an mtSSB knockout mouse model, but cannot support the switch to replication. Using deep sequencing as well as biochemical reconstitution experiments, we find that mtSSB is also necessary to restrict transcription initiation and primer formation to specific promoters and origins of replication both in vitro and in vivo. Pathological mutations in human mtSSB cannot efficiently support primer maturation and origin specific initiation of mtDNA replication in vitro.

2021-05-19 | PXD021799 | Pride

The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication

Project description:Single-stranded DNA (ssDNA) binding proteins protect regions of ssDNA formed during processes such as DNA replication and repair. We here devise a genetic screen and identify the mitochondrial ssDNA-binding protein (mtSSB) as a key regulator of mtDNA levels. In mitochondria, RNA synthesis from the light-strand promoter (LSP) is required for transcription as well as for generating the primers for initiation of mtDNA synthesis. We find that mtSSB is essential for mtDNA replication initiation, as transcription is strongly upregulated from the LSP in a mtSSB knockout mouse model, but cannot support the switch to replication. Using deep sequencing as well as biochemical reconstitution experiments, we find that mtSSB is also necessary to restrict transcription initiation and primer formation to specific promoters and origins of replication both in vitro and in vivo. Pathological mutations in human mtSSB cannot efficiently support primer maturation and origin specific initiation of mtDNA replication in vitro.

2022-08-29 | GSE159481 | GEO

The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication [sRNA]

2021-07-06 | GSE159570 | GEO

The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication [TruSeq]

2021-07-06 | GSE159569 | GEO

The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication

Project description:The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication

| PRJNA669705 | ENA

The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication [sRNA]

Project description:The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication [sRNA]

| PRJNA669760 | ENA

The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication [TruSeq]

Project description:The mitochondrial single-stranded DNA binding protein directs RNA primer formation for mtDNA replication [TruSeq]

| PRJNA669761 | ENA

POLRMT regulates the switch between replication-primer formation and gene expression of mammalian mtDNA

Project description:Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it remains highly controversial if POLRMT also serves as the primase for initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion and TFAM is thus protected from degradation of the AAA+ Lon protease in absence of POLRMT. Lastly, mitochondrial transcription elongation factor (TEFM) can compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role for this factor in transcription. In conclusion, we present here the first in vivo evidence that POLRMT has a key regulatory role in replication of mammalian mtDNA and is part of a mechanism that provides a switch between RNA primer formation for mtDNA replication and mtDNA expression. Isolated heart mitochondria from three control mice (L/L) and three Polrmt knockout mice (L/L, cre), aged 3-4 weeks, were sequenced and analyzed for differential expression.

2016-08-05 | E-GEOD-83368 | biostudies-arrayexpress

POLRMT regulates the switch between replication-primer formation and gene expression of mammalian mtDNA

2016-08-05 | GSE83368 | GEO

Mammalian RNase H1 directs RNA primer formation for mtDNA replication initiation and is also necessary for precise termination of mtDNA replication

Project description:The exact in vivo role for RNase H1 in mammalian mitochondria (mtDNA) has been much debated and we show here that it is essential for processing of RNA primers to provide site-specific initiation of mtDNA replication. Without RNase H1, mtDNA replication is instead initiated at non-canonical sites and becomes impaired. Furthermore, RNase H1 is also needed for replication completion and in its absence linear deleted mtDNA molecules extending between the two origins of mtDNA replication are formed. Finally, we report the first patient with a homozygous pathogenic mutation in the hybrid-binding domain (HBD) of RNase H1 causing impaired mtDNA replication. In contrast to catalytically dead pathological variants of RNase H1, this mutant version has enhanced enzyme activity. This finding shows that the RNase H1 activity must be strictly controlled to allow proper regulation of mtDNA replication.

2022-07-13 | GSE199198 | GEO

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