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In vivo composition of the mitochondrial nucleoid in mice

García-Villegas, Rodolfo ; Odenthal, Franka ; Giannoula, Yvonne ; Bonekamp, Nina A. ; Kühl, Inge ; Park, Chan Bae ; Spåhr, Henrik ; Motori, Elisa ; Levander, Fredrik LU orcid and Larsson, Nils Göran (2025) In Biochimica et Biophysica Acta - Molecular Cell Research 1872(6).
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 bacterial artificial chromosome (BAC) transgenic mice expressing FLAG-tagged TFAM protein (Tfam-FLAGBAC mice) to investigate the mt-nucleoid composition in vivo. Importantly, we show that the TFAM-FLAG protein is functional and complements the absence of the wild-type TFAM protein in homozygous Tfam knockout mice. We performed immunoprecipitation experiments from different mouse tissues and identified 12 proteins as core mt-nucleoid components by proteomics analysis. Among these, eight proteins correspond to mtDNA... (More)

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 bacterial artificial chromosome (BAC) transgenic mice expressing FLAG-tagged TFAM protein (Tfam-FLAGBAC mice) to investigate the mt-nucleoid composition in vivo. Importantly, we show that the TFAM-FLAG protein is functional and complements the absence of the wild-type TFAM protein in homozygous Tfam knockout mice. We performed immunoprecipitation experiments from different mouse tissues and identified 12 proteins as core mt-nucleoid components by proteomics analysis. Among these, eight proteins correspond to mtDNA replication and transcription factors, while the other four are involved in the mitoribosome assembly. In addition, we used the Tfam-FLAGBAC mice to identify ten 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 unraveling nine 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.

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author
; ; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Mitochondrial nucleoid, Mitochondrial translation, mtDNA expression, TFAM, Transgenic mice
in
Biochimica et Biophysica Acta - Molecular Cell Research
volume
1872
issue
6
article number
119955
publisher
Elsevier
external identifiers
  • scopus:105003570543
  • pmid:40246179
ISSN
0167-4889
DOI
10.1016/j.bbamcr.2025.119955
language
English
LU publication?
yes
id
21925091-124a-4b83-aa4e-6d4323cda4ed
date added to LUP
2025-07-18 08:27:58
date last changed
2025-07-18 08:28:52
@article{21925091-124a-4b83-aa4e-6d4323cda4ed,
  abstract     = {{<p>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 bacterial artificial chromosome (BAC) transgenic mice expressing FLAG-tagged TFAM protein (Tfam-FLAG<sup>BAC</sup> mice) to investigate the mt-nucleoid composition in vivo. Importantly, we show that the TFAM-FLAG protein is functional and complements the absence of the wild-type TFAM protein in homozygous Tfam knockout mice. We performed immunoprecipitation experiments from different mouse tissues and identified 12 proteins as core mt-nucleoid components by proteomics analysis. Among these, eight proteins correspond to mtDNA replication and transcription factors, while the other four are involved in the mitoribosome assembly. In addition, we used the Tfam-FLAG<sup>BAC</sup> mice to identify ten 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 unraveling nine interactors that could counteract the high TFAM levels to maintain active mtDNA transcription. Altogether, we demonstrate that the Tfam-FLAG<sup>BAC</sup> mice are a valuable tool for investigating the mt-nucleoid composition in vivo.</p>}},
  author       = {{García-Villegas, Rodolfo and Odenthal, Franka and Giannoula, Yvonne and Bonekamp, Nina A. and Kühl, Inge and Park, Chan Bae and Spåhr, Henrik and Motori, Elisa and Levander, Fredrik and Larsson, Nils Göran}},
  issn         = {{0167-4889}},
  keywords     = {{Mitochondrial nucleoid; Mitochondrial translation; mtDNA expression; TFAM; Transgenic mice}},
  language     = {{eng}},
  number       = {{6}},
  publisher    = {{Elsevier}},
  series       = {{Biochimica et Biophysica Acta - Molecular Cell Research}},
  title        = {{In vivo composition of the mitochondrial nucleoid in mice}},
  url          = {{http://dx.doi.org/10.1016/j.bbamcr.2025.119955}},
  doi          = {{10.1016/j.bbamcr.2025.119955}},
  volume       = {{1872}},
  year         = {{2025}},
}