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ESR 03 Project - Post Transcriptional regulation of mitochondrial gene expression by CLUH

Work Package 7: Identification of novel factor governing mitochondrial ribosomal assembly and protein synthesis.

Partner: Elena Rugarli

Institution: University of Cologne, Germany

Duration: 36 months

Objectives: The m-AAA protease is a large hexameric proteolytic complex in the inner mitochondrial membrane, composed of two subunits, AFG3L2 and SPG7/paraplegin. Mutations in the corresponding genes cause autosomal dominant spinocerebellar ataxia, SCA28, and autosomal recessive hereditary spastic paraplegia, respectively. Understanding the molecular function of the m-AAA protease is mandatory to unravel the pathogenesis of the human diseases and devise possible therapeutic strategies. The m-AAA protease regulates the turnover of inner membrane mitochondrial proteins such as respiratory complexes, and processes specific substrates, including the ribosomal component MRPL32. By studying a mouse model carrying a deletion in the Afg3l2 gene, we previously found that the levels of MRPL32 were drastically reduced, the mitochondrial ribosome was not completely assembled, and mitochondrial translation was impaired. This may underlie respiratory deficiencies in cells lacking Afg3l2 and explain neuronal death. This project aims to understand the molecular mechanisms by which the m-AAA protease affects assembly of the mitochondrial ribosome. To this end, our specific objectives are: (i) To investigate in a quantitative manner the proteome composition of the mitoribosome in the brain of mice lacking AFG3L2; (ii) To test the hypothesis that ribosomal assembly factors may be novel substrates of the m-AAA protease; (iii) To perform experiments to rescue mitochondrial translation in mouse embryonic fibroblasts lacking Afg3l2 by expressing MRPL32 and other identified substrates carrying artificial targeting sequences recognized by the mitochondrial matrix peptidase.

Expected results: Our project will shed light on the molecular mechanisms by which the m-AAA protease regulates mitochondrial translation. We will explore in detail the role of defective processing of MRPL32 in ribosome assembly, assess if other mitoribosomal proteins or ribosomal assembly factors are substrates of the protease, and evaluate the relevance of these findings by performing rescue experiments. The latter will be initially set up as proof of principle in mouse embryonic fibroblasts, but if successful will be translated in vivo by establishing appropriate mouse models. We expect that the data generated by this project will provide novel insights into the pathogenic mechanisms of neurodegeneration caused by m-AAA protease deficiencies. Moreover, our project will complement the project of ESR1 who is exploring how a mitochondrial matrix peptidase regulates mitochondrial translation, and the project of ERS2 that focuses on the prohibitin complex, which assembles in a supercomplex with the m-AAA protease.