12] Characterization of ribonuclease MRP function

Abstract

Publisher Summary Ribonuclease (RNase) MRP is a ribonucleoprotein endoribonuclease that cleaves an RNA sequence in a site-specific manner. The enzyme has two known RNA substrates: one in the mitochondria, where it processes a primer RNA for the initiation of DNA replication, and a second in the nucleus, where it processes the rRNA precursor to help generate the 5.8S rRNA. RNase MRP has been isolated from various organisms including human, mouse, rat, cow, Xenopus, yeast, Arabidopsis, and tobacco. The RNA component of the enzyme was found to be structurally and evolutionally related to RNase P RNA, the ribonucleoprotein endoribonuclease that processes the 5′ end of tRNA, and several protein components are shared between the two enzyme complexes. The data suggest that the enzymes, while ancient, have been highly conserved over time to perform essential cellular RNA-processing functions, both in the nucleus and the mitochondria. This chapter describes methodologies for testing various yeast mutants for endoribonuclease activity both in vivo and in vitro. In addition, a method for testing for assembly of the individual RNase MRP constituents with the complex is described.