CE12 - Génétique, génomique et ARN

Deciphering the role of the N-terminal extension of POLRMT in the regulation of mammalian mitochondrial gene expression – MitoPoNT

Submission summary

Mitochondrial dysfunction is associated to a broad spectrum of human diseases ranging from severe inherited metabolic disorders to common neurodegenerative diseases like Parkinson’s or Alzheimer’s disease, to heart disease, obesity, type 2 diabetes and cancer, and is also linked with common age- related diseases and the aging process itself. Mitochondria are indeed essential organelles found in every eukaryotic cell, providing cellular energy via their oxidative phosphorylation system (OXPHOS), which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Mitochondria have like the prokaryotic cells no compartmentalization between the different steps of mtDNA gene expression. Moreover, mitochondrial transcription and replication are linked processes, which both independently require the mammalian mitochondrial RNA polymerase (POLRMT). POLRMT thus fulfills key functions in building a functional OXPHOS system, however the role played in vivo by its large N-terminal extension (NTE) is still unknown. This NTE contains a domain with several pentatrico-peptide repeat (PPR) motifs. PPR-containing proteins are RNA-binding adaptor proteins that mediate interactions between RNA substrates and the enzymes that act on them. Interestingly, the crystal structure of POLRMT elongation complex suggests that newly synthesized RNAs exit toward the PPR motifs, but to date the function of the PPR motifs in the NTE remains unexplored. Moreover, several post-translational modification (PTM) sites, in particular phosphorylations, are predicted in silico in POLRMT, most of them locate in the NTE and some within the PPR-domain. These phosphorylation sites could, similar to the phosphorylation patterns in nuclear RNA polymerases, have an essential regulatory function, however, nothing is known about the PTMs in POLRMT and their in vivo role in mtDNA gene expression. Also, a couple of factors are proposed to regulate mtDNA gene expression by interacting with POLRMT to modulate its activity, but the underlying mechanisms are not understood and there is strong evidence that more POLRMT-interacting proteins remain to be identified.
In MitoPoNT we will shed light on the role of the enigmatic NTE of POLRMT, in particular on its PPR motifs, and aim at understanding how, through PTMs and interaction with other protein partners in trans, the NTE could modulate POLRMT function and mediate coupling between transcription and post-transcriptional steps of mtDNA expression.
Using mammalian cells, fission yeast and mouse tissues, MitoPoNT will focus on the POLRMT NTE to 1) conduct the first functional in vivo study of mammalian POLRMT PPR motifs by taking advantage of a yeast model and by generating variant cell lines through directed mutagenesis; 2) investigate the in vivo phosphorylation of POLRMT via an innovative targeted proteomics approach; 3) search for novel mammalian proteins interacting with POLRMT to regulate its function, using a mouse model.
These experiments will provide a deeper understanding of the molecular mechanism of action and of the regulatory aspects of POLRMT function, and will uncover partner proteins and their role in mitochondrial gene expression. In addition to its high fundamental importance, MitoPoNT is also undoubtedly relevant at the medical level, given the large number of human pathologies associated with mitochondrial dysfunctions including defects in POLRMT, as recently reported, and the potential of using POLRMT as a future therapeutic target to fight cancer. Thus, the data and knowledge gained from MitoPoNT will benefit to both basic and applied research and will have strong scientific and clinical impacts.

Project coordination

Inge KUHL (CNRS-Institut de Biologie Intégrative de la Cellule)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS-(I2BC) CNRS-Institut de Biologie Intégrative de la Cellule

Help of the ANR 336,522 euros
Beginning and duration of the scientific project: March 2021 - 48 Months

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