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ESR 01 Project - The Role of LONP1 in mitochondrial DNA maintenance

Work Package 5: Novel insights into maintenance of mitochondrial genome. 

Partner: Aleksandra Trifunovic

Institution: University of Cologne, Germany

Duration: 36 months

Objectives: CLPP (caseinolytic peptidase P) is a highly conserved serine protease present in bacteria and eukaryotic mitochondria. Loss-of-function mutations cause Perrault syndrome, a clinically heterogeneous autosomal-recessive condition characterized by sensorineural hearing loss, ovarian failure and shorter stature. In order to decipher the in vivo role of CLPP in mammals we have developed a CLPP - deficient mice (Clpp-/-). The CLPP - deficient mice faithfully recapitulate human syndrome. On the cellular level CLPP deficiency leads to an early decrease in Complex I activity, followed by a decrease in Complex IV activity later in life. Furthermore, we observed a deregulated mitochondrial transcript processing and a decrease in mitochondrial translation, which is compensated for by upregulation of de novo mitochondrial transcription. Search for CLPP interactors and substrates revealed a handful of proteins that are involved in RNAi processing like MRPP1 subunit of RNAse P and PNPT1. The main aim of this project is to decipher the role of CLPP in regulation of mitochondrial transcription. This will be achieved through pursue of the following objectives: (i) To further analyze and confirm candidates for substrates and interactors of CLPP involved in mitochondrial RNA processing and translation; (ii) To dissect molecular mechanism of action of CLPP substrates and interactors on mitochondrial mtRNA processing and function. Initial experiments will include in vitro CLPP reconstitution to directly test the degradation

efficiency of identified substrates. W. blot analysis of MRPP1 and PNPT1 steady state levels and their turnover rates in wild type and Clpp-/- cells will be performed. Physiological significance of detected changes will be evaluated upon normalization of MRPP1 and PNPT1 levels upon siRNA-mediated knockdown. The effect will be further evaluated using in organello transcription and translation assays. Northern blot analysis will be used to determine the levels of unprocessed and fully formed mtRNAs. Further experiments will depend on the outcome of these initial tests.

Expected results: We aim to identify the first mammalian CLPP substrate(s) and interactor(s) and decipher their physiological role. Our preliminary screens have revealed a group of proteins involved in a fine-tuning of mitochondrial transcription through coordination of mtRNA processing. By combining in vivo and in vitro methods we will make a catalog of CLPP interactors and substrates. We expect to identify molecular mechanism and specific CLPP substrates that modulate mtRNA processing.