Characterization and Purification of Saccharomyces cerevisiae RNase MRP Reveals a New Unique Protein Component

Abstract

In the yeast Saccharomyces cerevisiae, RNase mitochondrial RNA processing (MRP) is an essential endoribonuclease that consists of one RNA component and at least nine protein components. Characterization of the complex is complicated by the fact that eight of the known protein components are shared with a related endoribonuclease, RNase P. To fully characterize the RNase MRP complex, we purified it to apparent homogeneity in a highly active state using tandem affinity purification. In addition to the nine known protein components, both Rpr2 and a protein encoded by the essential gene YLR145w were present in our preparations of RNase MRP. Precipitation of a tagged version of Ylr145w brought with it the RNase MRP RNA, but not the RNase P RNA. A temperature-sensitive ylr145w mutant was generated and found to exhibit a rRNA processing defect identical to that seen in other RNase MRP mutants, whereas no defect in tRNA processing was observed. Homologues of the Ylr145w protein were found in most yeasts, fungi, and Arabidopsis. Based on this evidence, we propose that YLR145w encodes a novel protein component of RNase MRP, but not RNase P. We recommend that this gene be designated RMP1, for RNase MRP protein 1. RNase MRP1 is a highly conserved and essential ribonucleoprotein endoribonuclease that cleaves substrates in at least two intracellular compartments. Most RNase MRP is localized to the nucleolus (1), where a role in processing of rRNA precursors has been identified (2). RNase MRP-mediated cleavage at the A 3 site of pre-rRNA ultimately leads to the generation of 5.8S(S) rRNA (3-5). A fraction of RNase MRP RNA finds its way to the mitochondrion (6, 7). Mitochondrial RNase MRP processes RNA transcripts, which serve as primers for the leading strand of mitochondrial DNA replication (8, 9). Recent data also support a role for RNase MRP mRNA degradation, whereby cleavage of the 5Ј-untranslated region (UTR) of CLB2 mRNA, which encodes a B-type cyclin, leads to its rapid degradation and aids cell cycle progression (10).In Saccharomyces cerevisiae, all the known components of RNase MRP are essential for viability. To date, a single RNA component and at least nine protein components of nuclear RNase MRP have been identified. The subunit composition of RNase MRP closely resembles that of a related ribonucleoprotein endoribonuclease, RNase P, which processes tRNA precursors to generate mature 5Ј termini. Eight of the proteins associated with RNase MRP (Pop1, Pop3, Pop4, Pop5, Pop6, Pop7, Pop8, and Rpp1) are also components of RNase P (11-14). An RNA-binding protein, encoded by the gene SNM1, is the only known protein component that associates with RNase MRP RNA but not RNase P RNA (15). Similarly, Rpr2p has been identified as a unique protein component of the RNase P complex (14).The similarities between RNase MRP and RNase P extend beyond that of shared protein components. The RNA subunits of RNase MRP and RNase P are evolutionarily and structurally related (16,17). They share only weak sequence homology, but they fold into...

Interactions of a Pop5/Rpp1 heterodimer with the catalytic domain of RNase MRP

“…RNase MRP (as a z1:1 mix with RNase P) was purified from S. cerevisiae strains EK-Pop5 and EK-Rpp1 using tandem affinity purification (Rigaut et al 1999) and following a protocol described in Salinas et al (2005) and Esakova et al (2008). Strains EK-Pop5 and EK-Rpp1 were based on yeast strain YSW1 ), a generous gift from Mark Schmitt.…”

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA–protein interactions

“…All of these proteins, except for Rpr2, are also found in RNase MRP (Chamberlain et al 1998). Yeast RNase MRP has 10 known protein subunits: in addition to proteins found in RNase P, RNase MRP has two essential proteins: Snm1 (Schmitt and Clayton 1994) and Rmp1 (Salinas et al 2005).…”

Functional characterization of the conserved amino acids in Pop1p, the largest common protein subunit of yeast RNases P and MRP

“…Eight proteins are the same as those in yeast RNase P: Pop1p, Pop3p, Pop4p, Pop5p, Pop6p, Pop7p, Pop8p, and Rpp1p (Chamberlain et al 1998 and references therein). The protein subunit unique to RNase P is the Rpr2p, whereas Snm1p and Rmp1p are specific to RNase MRP (Schmitt and Clayton 1994;Chamberlain et al 1998;Salinas et al 2005). NME1 RNA and the RPR1 RNA conform to similar secondary structures and have several small patches of conserved sequence found in all RNase P RNAs (Forster and Altman 1990;Frank et al 2000;Li et al 2002).…”

“…Recently, an additional role for RNase MRP in cell cycle progression in yeast through selective mRNA turnover has been reported (Gill et al 2004). The yeast RNase MRP holoenzyme consists of one RNA subunit, NME1 RNA, and at least 10 protein subunits (Schmitt and Clayton 1994;Chamberlain et al 1998;Salinas et al 2005). Eight proteins are the same as those in yeast RNase P: Pop1p, Pop3p, Pop4p, Pop5p, Pop6p, Pop7p, Pop8p, and Rpp1p (Chamberlain et al 1998 and references therein).…”