Characterization of human mitochondrial Ribonuclease P

Abstract

Sequences preceding the 5' terminus of tRNAs in the primary transcript are removed by an ubiquitous endonuclease called Ribonuclase P (RNase P). Since all RNase Ps characterized so far contain a catalytically active RNA component it is widely assumed that RNase P is an ubiquitous ribozyme. Studies showing that organellar RNase P of higher eukaryotes are RNA-free, protein-only enzymes were met with great scepticism. To finally resolve this controversy, components of human mitochondrial RNase P were identified using a novel approach combining partial purifications with shot-gun proteomics. Candidates identified were screened by expression in human cell lines for their association with mitochondrial RNase P activity. Human mitochondrial RNase P turned out to be composed of 3 proteins and no RNA. Using bacterial expressed, affinity-purified proteins we functionally reconstituted the enzyme thereby demonstrating that no trans-acting RNA component is required for human mitochondrial RNase P activity. None of the protein subunits is homologous to any known RNase P protein. Instead, the enzyme appears to be a novel type of patchwork enzyme, composed of proteins recruited from, and still involved in at least 2 different biochemical pathways; tRNA-Methylation and fatty acid metabolism. This is the first proven example of a "non-ribozymal" RNase P and finally lays the discussion to a rest which lasted for 2 decades about the existence of an RNA-free tRNA 5' processing enzyme.<br />