Characterization of human mitochondrial ribonuclease P

tRNA molecules, the universal adapters between mRNA and protein, are synthesized as precursors that have to undergo a variety of maturation steps. One of these steps, the removal of 5` extensions, is generally carried out by an endonuclease called ribonuclease P (RNase P). All RNase P enzymes that have been characterized in detail, are ribonucleoproteins, i.e., they consist of RNA and protein. The RNA of bacterial RNase P alone is catalytically active in vitro, and the RNA component appears to be the catalytical subunit of other RNase P enzymes as well. However, evidence has accumulated that some organellar RNase P enzymes may consist of protein only. Human mitochondrial RNase P is of protein-like density, is insensitive to nuclease digestion, and no copurifying RNA has been identified. This project aims to identify the protein component(s) of human mitochondrial RNase P using a combination of classical protein purification methods and 2 D electrophoresis. The enzyme will be partially purified employing various strategies in parallel, in a small, semi-preparative scale. The components of these batches of mitochondrial RNase P activity, which have been partially purified following different routes, will subsequently be analyzed by 2 D electrophoresis. Thereby a subset of matching proteins, reproducibly copurifying with mitochondrial RNase P activity irrespective of the employed purification strategy, shall be identified. Matching, copurifying proteins will be subjected to peptide identification by mass spectrometry and subsequent genetic analysis. Candidate genes will be silenced by RNA interference. Knock-out of putative mitochondrial RNase P genes is expected to lead to the accumulation of mitochondrial tRNA precursors or processing intermediates, or to a drop in tRNA steady-state levels. To finally verify a protein as a mitochondrial RNase P protein, epitope-tagged versions of candidates will be expressed in human cells to generate a tagged form of mitochondrial RNase P potentially susceptible to immuno-depletion from mitochondrial extracts. Selection of a protein by this procedure will most strongly support its role as a mitochondrial RNase P component.

tRNA molecules, the universal adapters between mRNA and protein, are synthesized as precursors that have to undergo a variety of maturation steps. One of these steps, the removal of 5` extensions, is generally carried out by an endonuclease called ribonuclease P (RNase P). All RNase P enzymes that have been characterized in detail, are ribonucleoproteins, i.e., they consist of RNA and protein. The RNA of bacterial RNase P alone is catalytically active in vitro, and the RNA component appears to be the catalytical subunit of other RNase P enzymes as well. However, evidence has accumulated that some organellar RNase P enzymes may consist of protein only. Human mitochondrial RNase P is of protein-like density, is insensitive to nuclease digestion, and no copurifying RNA has been identified. This project aims to identify the protein component(s) of human mitochondrial RNase P using a combination of classical protein purification methods and 2 D electrophoresis. The enzyme will be partially purified employing various strategies in parallel, in a small, semi-preparative scale. The components of these batches of mitochondrial RNase P activity, which have been partially purified following different routes, will subsequently be analyzed by 2 D electrophoresis. Thereby a subset of matching proteins, reproducibly copurifying with mitochondrial RNase P activity irrespective of the employed purification strategy, shall be identified. Matching, copurifying proteins will be subjected to peptide identification by mass spectrometry and subsequent genetic analysis. Candidate genes will be silenced by RNA interference. Knock-out of putative mitochondrial RNase P genes is expected to lead to the accumulation of mitochondrial tRNA precursors or processing intermediates, or to a drop in tRNA steady-state levels. To finally verify a protein as a mitochondrial RNase P protein, epitope-tagged versions of candidates will be expressed in human cells to generate a tagged form of mitochondrial RNase P potentially susceptible to immuno-depletion from mitochondrial extracts. Selection of a protein by this procedure will most strongly support its role as a mitochondrial RNase P component.