Substrate Recognition by Human Mitochondrial RNase P: Moonlighting of a tRNA Methyltransferase

Ribonuclease P (RNase P) catalyzes removal of 5' end leader sequences from precursor tRNA (pre-tRNA). In most organisms, RNase P is a ribonucleoprotein complex with a catalytic RNA subunit. However, a recently discovered human mitochondrial RNase P (mtRNase P) is composed solely of three protein subunits: MRPP1, 2 and 3. The MRPP1•MRPP2 subcomplex contains m1N9 tRNA methyltransferase (MRPP1), while MRPP3 belongs to a new class of metallonucleases. We reconstituted human mtRNase P proteins in vitro and measured reactivity with different types of pre-tRNA substrate to study substrate recognition. For a canonical pre-tRNA, transient kinetic measurements show that the MRPP1•MRPP2 subcomplex activates the endonuclease activity of MRPP3 by about 2000-fold and enhances substrate affinity by 50-fold. For a non-canonical human mt-pre-tRNA, significant miscleavage was observed with MRPP3 alone but the MRPP1•MRPP2 subcomplex rescued miscleavage and enhanced the cleavage rate constant by 1000-fold. In addition, in vi...