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ESR 11 Project - Mitochondrial RNA granule associated proteins: investigating their roles in mtDNA gene regulation and expression

Work Package 6: Molecular mechanisms of mitochondrial transcription and RNA processing.

Partner: Jean-Claude Martinou

Institution: University of Geneva, Switzerland

Duration: 36 months

Objectives: To investigate the mechanism of action of FASTK family members and their role in mitochondrial gene expression.

Expected results: Mitochondrial gene expression is currently poorly understood. Mitochondrial RNAs are transcribed from only three promoters into large polycistronic transcripts that are then processed, largely by removal of tRNAs according to the “tRNA punctuation model”, to yield individual RNAs. Despite originating from the same polycistronic transcripts, levels of each RNA species vary considerably. Furthermore, the mechanism by which the individual RNAs are released from the polycistronic transcript is not fully explained by the tRNA punctuation model, as there are a number of atypical junctions which lack flanking tRNAs including (1) the 5’ end of CO1 mRNA; (2) the junction between the ATP8/6 and CO3 mRNAs; (3) the junction between the ND5 and CYB mRNAs; and (4) the 3’ end of ND6 mRNA. We have recently observed that mitochondrial RNA processing, at least in part, takes place in a new type of RNA granules in the mitochondrial matrix of mammalian cells. Amongst the components of the RNA granules involved in this process, are members of the FASTK family. This family is composed of six members, FASTK, which is both cytosolic and mitochondrial, and FASKD1-5 which all are mitochondrial. We have found that FASTK and FASTKD2 are involved in the stability of ND6 mRNA and that FASTKD4 and FASTKD1 antagonistically regulate expression of a number of other mRNAs. Moreover, we have evidence that, through their RAP domain, these proteins may act as endonucleases. In this project, we propose to identify novel components of the mitochondria RNA granules using in particular the BioID approach described by Roux et al. (JCB, 2012). Moreover, we propose to identify MRG assembly factors using a functional approach based on large-scale gene inactivation, coupled with a state-of-art high content imaging analysis of MRGs. This approach will allow us potentially to identify genes that lead to a wide spectrum of morphological changes in MRGs. The candidate genes emerging from the first library screening will be confirmed in secondary assays, and further characterized in later stages of the Project. We will also identify the RNA targets of each FASTK family member using an approach recently described by Lapointe et al. (Nature Methods, 2015), consisting in the fusion of the protein of interest with a poly(U) polymerase that labels the target RNAs with polyUs. Once the RNA targets of these proteins identified, we will develop an acellular assay to test the hypothesis that members of the family display an endonucleolytic activity.