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Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA [RNA-seq]

ABSTRACT: Cellular metabolism relies on the regulation and maintenance of mitochondrial DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist in each cell, yet how mtDNA is packaged into individual mitochondrial nucleoids remains poorly resolved, as mitochondria lack histones or other chromatin machinery important for nuclear genome compaction. Here, we used long-read single-molecule accessibility mapping to resolve the compaction of individual full-length mitochondrial nucleoids at single-nucleotide resolution. We find that, unlike the nuclear genome, human mtDNA largely undergoes an all-or-none global compaction, with the majority of nucleoids existing in an inaccessible, inactive state. Highly accessible mitochondrial nucleoids are occupied by transcription and replication machinery, and selectively form a D-loop structure. In addition, we demonstrate that the primary nucleoid-associated protein TFAM directly modulates the fraction of inaccessible nucleoids both in vivo and in vitro, and acts via a ‘nucleation and spreading’ mechanism to coat and compact mitochondrial nucleoids. Overall, these findings expose the primary architecture of mtDNA packaging and regulation in humans.

ORGANISM(S): Homo sapiens

PROVIDER: GSE240902 | GEO | 2023/11/15

REPOSITORIES: GEO

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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.

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Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA [RNA-seq]

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