MtDNA breaks compromise mitochondrial membrane ultrastructure and trigger an integrated stress response
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA885786 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21765600_1.fastq.gz | Fastqsanger.gz | |||
| SRR21765600_2.fastq.gz | Fastqsanger.gz | |||
| SRR21765601_1.fastq.gz | Fastqsanger.gz | |||
| SRR21765601_2.fastq.gz | Fastqsanger.gz | |||
| SRR21765602_1.fastq.gz | Fastqsanger.gz |
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Project description:Mitochondrial DNA (mtDNA) breaks are deleterious lesions that lead to degradation of mitochondrial genomes and subsequent reduction in mtDNA copy number. The signaling pathways activated in response to mtDNA damage remain ill-defined. Using mitochondrial targeted restriction enzymes, we show that cells with mtDNA breaks exhibit reduced respiratory complexes, loss of membrane potential, and mitochondrial protein import defect. Furthermore, mtDNA damage activates the integrated stress response (ISR) through phosphorylation of eIF2α by the OMA1-DELE1-HRI pathway. Electron microscopy reveals concomitant defects in mitochondrial membranes and cristae ultrastructure. Notably, inhibition of the ISR exacerbates mitochondrial defects and delays the recovery of mtDNA copy number, thereby implicating this stress response in mitigating mitochondrial dysfunction following mtDNA damage. Last, we provide evidence suggesting that ATAD3A, a membrane-anchored protein that interacts with nucleoids, relays the signal from mtDNA breaks to the inner mitochondrial membrane. Altogether, our study fully delineates the sequence of events linking damaged mitochondrial genomes with the cytoplasm and uncovers an unanticipated role for the ISR in response to mitochondrial genome instability.
2022-10-04 | GSE214512 | GEO
Project description:Mitochondrial DNA (mtDNA) breaks are toxic lesions that lead to degradation of mitochondrial genomes and subsequent reduction in mtDNA copy number. The signaling pathways activated in response to mtDNA damage remain poorly understood. We reasoned that factors that sense mtDNA damage are likely to be recruited to nucleoids shortly after break formation, prompting us to develop a proteomic-based approach to survey the interactome of mitochondrial nucleoids. Specifically, we combined proximity-dependent biotin labeling with Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and tandem mass spectrometry. We used mitochondrial targeted restriction enzymes for the proximity probe and show that cells with mtDNA breaks exhibit reduced respiratory complexes, loss of membrane potential, and mitochondrial protein import defect. Notably, mtDNA breaks activate the integrated stress response (ISR) through phosphorylation of eIF2a by the OMA1-DELE1-HRI pathway. Inhibition of the ISR exacerbates mitochondrial defects and delays the recovery of mtDNA copy number, hinting at a role for the ISR in mitigating mitochondrial dysfunction following mtDNA damage. Electron microscopy reveals defective mitochondrial membranes and cristae ultrastructure. Last, we highlight a potential role for ATAD3A - a protein that is anchored to the inner mitochondrial membrane while interacting with nucleoids - in relaying the signal from damaged mtDNA to the mitochondrial inner membrane. Altogether, our study delineates the sequence of events linking damaged mitochondrial genomes to the cytoplasmic stress response. The mass spectrometry raw files for the combined SILAC and proximity-dependent biotin labeling experiment are deposited here.
2022-10-05 | MSV000090459 | MassIVE
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Project description:Mitochondria serve as the cellular powerhouse, and their distinct DNA makes them a prospective target for gene editing to treat genetic disorders. However, the impact of genome editing on mitochondrial DNA (mtDNA) stability remains a mystery. Our study reveals previously unknown risks of genome editing that both nuclear and mitochondrial editing cause broad transfer of mitochondrial DNA segments into the nuclear genome in various cell types including human cell lines, primary T cells, and mouse embryos. Furthermore, drug-induced mitochondrial stresses and mtDNA breaks exacerbate this transfer of mtDNA into the nuclear genome. Notably, we observe that mitochondrial targeting editors, mitoTALEN and recently developed base editor DdCBE, can also induce widespread mtDNA integrations. However, we provide a practical solution to suppress mtDNA transfer by co-expressing TREX1 or TREX2 exonucleases during DdCBE editing. These findings also shed light on the origins of mitochondrial-nuclear DNA segments.
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Project description:Integrity of mitochondrial DNA (mtDNA), encoding several subunits of the respiratory chain, is essential to maintain mitochondrial fitness. Mitochondria, as a central hub for metabolism, are affected in a wide variety of human diseases but also during normal ageing, where mtDNA integrity is compromised. Mitochondrial quality control mechanisms work at different levels, and mitophagy and its variants are critical to remove dysfunctional mitochondria and mtDNA to maintain cellular homeostasis. Understanding the mechanisms governing a selective turnover of mutation-bearing mtDNA without affecting the entire mitochondrial pool is fundamental to design therapeutic strategies against mtDNA diseases and ageing. Here, we show that mtDNA damage after expressing a dominant negative version of the mitochondrial helicase Twinkle, or by chemical means, leads to an exacerbated mtDNA turnover. mtDNA removal depends on lysosomal function and requires the autophagy protein Atg5 but is independent of canonical mitophagy or autophagy. Using proximity labelling, we demonstrated that the area of influence of mitochondrial nucleoids differs upon mtDNA damage, which induces mitochondrial membrane remodelling and endosomal recruitment in close proximity to mitochondrial nucleoid sub compartments. Targeting of nucleoids is controlled by the mitochondrial transmembrane proteins ATAD3 and SAMM50, which together with the endosomal trafficking protein VPS35, orchestrate endosomal nucleoid engulfment. SAMM50 acts as a gatekeeper to avoid mtDNA release to the cytoplasm and facilitating mtDNA transfer to VPS35. Lastly, we show that stimulation of lysosomal activity by rapamycin selectively removes mtDNA deletions in vivo, without affecting mtDNA copy number. With these results, we unveil the molecular players of a new complex mechanism specifically targeting and removing mutant mtDNA which occurs outside the mitochondrial network, a process with multiple potential benefits to understand human mtDNA related diseases, either inherited, acquired or due to normal ageing.
2022-11-07 | PXD023939 | Pride
Project description:DNA damage activates a robust transcriptional stress response, but much less is known about how DNA damage impacts translation. The advent of genome editing with Cas9 has intensified interest in understanding cellular responses to DNA damage. Here, we find that DNA double-strand breaks (DSBs), including those induced by Cas9, trigger the loss of ribosomal protein RPS27A from ribosomes via p53-independent proteasomal degradation. Comparisons of Cas9 and dCas9 ribosome profiling and mRNA-seq experiments reveal a global translational response to DSBs that precedes changes in transcript abundance. Our results demonstrate that even a single DSB can lead to altered translational output and ribosome remodeling, suggesting caution in interpreting cellular phenotypes measured immediately after genome editing.
2021-11-15 | GSE122615 | GEO
Project description:Mitochondria serve as the cellular powerhouse, and their distinct DNA makes them a prospective target for gene editing to treat genetic disorders. However, the impact of genome editing on mitochondrial DNA (mtDNA) stability remains a mystery. Our study reveals previously unknown risks of genome editing that both nuclear and mitochondrial editing cause discernible transfer of mitochondrial DNA segments into the nuclear genome in various cell types including human cell lines, primary T cells, and mouse embryos. Furthermore, drug-induced mitochondrial stresses and mtDNA breaks exacerbate this transfer of mtDNA into the nuclear genome. Notably, we observe that mitochondrial editors, including mitoTALEN and recently developed base editor DdCBE, can also enhance crosstalk between mtDNA and the nuclear genome. Moreover, we provide a practical solution by co-expressing TREX1 or TREX2 exonucleases during DdCBE editing. These findings imply genome instability of mitochondria during induced DNA breaks and explain the origins of mitochondrial-nuclear DNA segments.
2024-09-24 | GSE272364 | GEO
Project description:Mitochondria serve as the cellular powerhouse, and their distinct DNA makes them a prospective target for gene editing to treat genetic disorders. However, the impact of genome editing on mitochondrial DNA (mtDNA) stability remains a mystery. Our study reveals previously unknown risks of genome editing that both nuclear and mitochondrial editing cause discernible transfer of mitochondrial DNA segments into the nuclear genome in various cell types including human cell lines, primary T cells, and mouse embryos. Furthermore, drug-induced mitochondrial stresses and mtDNA breaks exacerbate this transfer of mtDNA into the nuclear genome. Notably, we observe that mitochondrial editors, including mitoTALEN and recently developed base editor DdCBE, can also enhance crosstalk between mtDNA and the nuclear genome. Moreover, we provide a practical solution by co-expressing TREX1 or TREX2 exonucleases during DdCBE editing. These findings imply genome instability of mitochondria during induced DNA breaks and explain the origins of mitochondrial-nuclear DNA segments.
2024-09-24 | GSE272360 | GEO
Project description:Transfer of mitochondrial DNA into the nuclear genome during gene editing [PEM-Seq mtDNA breaks]
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