ESR 10 Project - Biogenesis and function of mitochondrial ribosome
Work Package 7: Identification of novel factor governing mitochondrial ribosomal assembly and protein synthesis.
Partner: Michal Minczuk
Institution: MRC-Mitochondrial Biology Unit, Cambridge, UK
Duration: 36 months
Objectives: The key objective of the proposed research is to study nuclear-encoded proteins involved in mitochondrial RNA (mt-RNA) metabolism in order to understand how their dysfunction is linked with human mitochondrial pathologies. Post-transcriptional control of RNA stability, processing, nucleotide modification, and degradation is key to the regulation of gene expression in all living cells. In mitochondria, these post-transcriptional processes are also vital for proper expression of the thirteen proteins encoded by the mitochondrial genome, through ensuring a proper function of mitochondrial mRNAs, tRNAs and rRNAs. However, our knowledge on mt-RNA metabolic pathways, especially in the context of their tissue-specific regulatory role, is far from complete. All of the proteins involved in mt-RNA metabolism are encoded by the nucleus, and must be imported into the organelle. Importantly, defects in the expression of mtDNA-encoded genes due to nuclear DNA mutations often lead to deficiencies in cellular energy provision and represent a rapidly growing group of human disorders. In more detailed terms, three methyltransfereses (MTases) involved in epitranscriptomic modification of mitochondrial transfer RNA (mt-tRNA) and ribosomal RNA (mt-rRNA) with identified mutations in patients affected by mitochondrial diseases will be studied. Using recently developed, high-throughput methods (miCLIP, bisulphite RNASeq) and, a more standard, reverse transcription primer extension (RT-PEx) analysis, we have already unravelled target mt-RNA nucleotides for two of these MTases upon gene inactivation by RNAi. As a continuation of these studies, the proposed project will involve: (i) Comprehensive analysis of the mitochondrial transcriptome and its post-transcriptional RNA methylation in patient-derived cells; (ii) Analysing mitochondrial translation (by 35S-methionine labelling and RNASeq-based mitoribosome profiling) and mitochondrial ribosome integrity (by mass spectrometry complexome profiling upon sucrose gradient sedimentation or BN-PAGE) in patient-derived cells; (iii) Study expression levels of the identified MTases and the levels of corresponding mt-RNA modifications across human tissues; (iv) Rescuing of pathological molecular phenotype in patient-derived cells by re-expression (upon lentiviral transduction) of wild-type copy cDNA of specific MTases; (v) In vitro study of biochemical properties of recombinant proteins and their pathogenic mutants; (vi) Identifying other mitochondrial proteins that potentially interact with MTases (by pull-down of tagged proteins followed by SILAC).
Expected results: In general terms, the project will provide new knowledge about how post-transcriptional mt-RNA modifications contribute to mitochondrial gene expression and the progression of mitochondrial diseases. More specifically, proposed study on disease-associated variants will be helpful in: defining of a full catalogue of genes underlying mitochondrial disease, unrevealing new molecular details of the processes of mitochondrial gene expression and its regulation and understanding the tissue- and cellspecificity observed in mitochondrial disease.