Function and Inhibition of Protein-only Ribonuclease P

Abstract

Ribonuclease P (RNase P) is metal-dependent endonuclease responsible for the removal of extraneous 5’-ends in precursor tRNA (pre-tRNA). RNase P is essential and present in all domains of life. In many organisms, RNase P is a ribonucleoprotein (RNP) complex with a catalytic RNA subunit and accessory proteins. Our view of RNase P expanded with the identification of protein-only RNase P (PRORP) found in plants and human mitochondria. In plants, PRORP endonuclease activity functions as a single-subunit enzyme. In contrast, human mitochondrial RNase P (mtRNase P) is composed of three protein subunits: mitochondrial RNase P protein (MRPP) 1, 2 and 3. MRPP3, which is homologous to plant PRORP, contains the active site that catalyzes pre-tRNA hydrolysis. However, MRPP3 requires a tRNA methyltransferase (MRPP1) and a dehydrogenase/reductase (MRPP2) for catalysis. To investigate the mechanism of pre-tRNA cleavage catalyzed by human mitochondrial RNase P, we describe a model for pre-tRNA hydrolysis that highlights the importance of the MRPP1/2 subcomplex on recognition and processing of mitochondrial tRNA transcripts. Our data suggests that pre-tRNA stabilizes the interactions between MRPP3 and MRPP1/2. We propose that binding of MRPP1/2 to pre-tRNA alters the pre-tRNA structure, yielding a pre-tRNA structure that is recognized by MRPP3 at a higher affinity and is efficiently cleaved. Furthermore, the identification of inhibitors against PRORP1 from a high throughput screen provides lead compounds to probe PRORP activity. We have identified an uncompetitive and a time-dependent inhibitor of PRORP1, revealing several cysteine residues that may be involved in the inactivation of PRORP1. Both compounds inhibit A. thaliana PRORP1 and human MRPP1/2/3. Taken together, this work provides a mechanistic approach for investigating the function of PRORP in the human mitochondria and multi-subunit RNA-protein complexes and a framework for studying mitochondrial disease states that arise from dysfunctional RNA processing.