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Definition of the composition of the mitochondrial nucleoid in mice

ABSTRACT: Mitochondrial DNA (mtDNA) is compacted into dynamic structures called mitochondrial nucleoids (mt-nucleoids), with the mitochondrial transcription factor A (TFAM) as the core packaging protein. We generated Tfam-FLAGBAC mice to investigate the mt-nucleoid composition in vivo. Importantly, we showed that the TFAM-FLAG protein is functional and complements the absence of the wild-type TFAM. We identified proteins as core mt-nucleoid components by proteomics analysis of Tfam-FLAGBAC mice. Surprisingly, the mitoribosome subunit MRPS38 was part of the mt-nucleoid, and its downregulation led to the increase of mtDNA replication and transcription. Thus, MRPS38 might have a novel function repressing mtDNA expression. Also, we identified proteins that may stabilize TFAM-FLAG upon depletion of the mitochondrial RNA polymerase despite the absence of mtDNA and induction of the LONP1 protease. Finally, we evaluated the changes in mt-nucleoids caused by very high levels of TFAM-FLAG and discovered eight new interactors that could counteract the high TFAM levels to maintain active mtDNA transcription. Altogether, we demonstrate that the Tfam-FLAGBAC mice are a valuable tool for investigating the mt-nucleoid composition in vivo.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart, Brain, Skeletal Muscle, Liver, Colon, Kidney

SUBMITTER: Fredrik Levander

LAB HEAD: Nils-GÃ¶ran Larsson

PROVIDER: PXD047950 | Pride | 2025-09-23

REPOSITORIES: Pride

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			Action	DRS
	20200901_HF2_01_Rodolfo_Agarose_200626_K4-2_.raw	Raw
	20200901_HF2_05_Rodolfo_Agarose_200626_C3-4_.raw	Raw
	20200901_HF2_08_Rodolfo_Agarose_200626_B4-1_.raw	Raw
	20200901_HF2_09_Rodolfo_Agarose_200626_H3-3_.raw	Raw
	20200901_HF2_10_Rodolfo_Agarose_200626_C1-4_.raw	Raw

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Project description:The goal of this analysis was to utilize microarray profiling to identify basal alterations in gene expression in response to TFAM depletion and mtDNA stress. Mitochondrial DNA (mtDNA) is normally present at thousands of copies per cell and is packaged into several hundred higher-order structures termed nucleoids. The abundant mtDNA-binding protein, TFAM (transcription factor A,mitochondrial), regulates nucleoid architecture, abundance and segregation. Complete mtDNA depletion profoundly impairs oxidative phosphorylation, triggering calcium-dependent stress signalling and adaptive metabolic responses. However, the cellular responses to mtDNA instability, a physiologically relevant stress observed in many human diseases and ageing, remain poorly defined. Here we show that moderate mtDNA stress elicited by TFAM deficiency engages cytosolic antiviral signalling to enhance the expression of a subset of interferon-stimulated genes. Mechanistically, we find that aberrant mtDNA packaging promotes escape of mtDNA into the cytosol, where it engages the DNA sensor cGAS (also known as MB21D1) and promotes STING (also known as TMEM173)–IRF3-dependent signalling to elevate interferon-stimulated gene expression, potentiate type I interferon responses and confer broad viral resistance. Furthermore, we demonstrate that herpesviruses induce mtDNA stress, which enhances antiviral signalling and type I interferon responses during infection. Our results further demonstrate that mitochondria are central participants in innate immunity, identify mtDNA stress as a cell-intrinsic trigger of antiviral signaling and suggest that cellular monitoring of mtDNA homeostasis cooperates with canonical virus sensing mechanisms to fully engage antiviral innate immunity. Murine embryonic fibroblasts were isolated from wild-type or Tfam+/- E13.5 littermate embryos. RNA from passage-matched wild-type and Tfam+/- MEF lines was extracted in duplicate and hybridized onto Affymetrix microarrays. Four arrays were performed in total with two technical replicates per genotype.

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Definition of the composition of the mitochondrial nucleoid in mice

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