Composition and characterization of nuclear ribonuclease P from Saccharomyces cerevisiae.

Abstract

Ribonuclease P (RNase P) is the enzyme responsible for 5<super>' </super> processing of precursor tRNA (pre-tRNA) molecules. The holoenzyme is composed of both RNA and protein subunits. Bacterial RNase P has been studied extensively and the RNA subunits have been shown to be catalytic. The single RNase P protein subunit from bacterial enzymes is required for function <italic> in vivo</italic>. The eukaryotic nuclear RNase P RNA subunit has been identified and analyzed, but purification of the protein component of the RNase P holoenzyme has proven difficult due to its lability and low abundance. Characterization of RNase P was initiated with analysis of an RNA subunit mutation. The yeast strain harboring the RNA mutation displayed defects in precursor rRNA (pre-rRNA) as well as pre-tRNA processing. This analysis of a known RNase P subunit confirmed a close evolutionary and functional relationship between RNase P and RNase MRP, an endoribonuclease that is involved in pre-rRNA processing. To address the question of what proteins were necessary for RNase P function, the RNase P holoenzyme was purified using ion-exchange chromatography and density gradient sedimentation. Identification of a large complex of at least nine proteins in association with RNase P activity was obtained by mass spectrometry methods. Proteins found among these include four candidate RNase P subunits identified by genetic methods intended to find RNase MRP components. Five other proteins were products of hypothetical open reading frames in the <italic> S. cerevisiae</italic> genome. Employing genetic methods available in yeast, future efforts will focus on determining the role of these proteins in pre-tRNA processing, pre-rRNA processing, and other possible cellular functions.