Proc. Natl. Acad. Sci. USA Vol. 95, pp. 6716–6721, June 1998 Biochemistry Rpp2, an essential protein subunit of nuclear RNase P, is required for processing of precursor tRNAs and 35S precursor rRNA in Saccharomyces cerevisiae VIKTOR STOLC*, ALEXANDER KATZ†, AND SIDNEY ALTMAN†‡ †Department of Biology, Yale University, New Haven, CT 06520; and *Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510 Contributed by Sidney Altman, March 31, 1998 ABSTRACT RPP2, an essential gene that encodes a 15.8- has been suggested that RNase P is an ancestor of RNase kDa protein subunit of nuclear RNase P, has been identified MRP, because RNase MRP has been found only in eukaryotes in the genome of Saccharomyces cerevisiae. Rpp2 was detected (20). Yeast RNase MRP functions in processing of precursor by sequence similarity with a human protein, Rpp20, which rRNAs at the A3 site in the internal transcribed sequence of copurifies with human RNase P. Epitope-tagged Rpp2 can be the 35S precursor rRNA (21) and also cleaves RNA primers found in association with both RNase P and RNase mitochon- for mitochondrial DNA replication (22, 23). Although RNase drial RNA processing in immunoprecipitates from crude MRP does not cleave ptRNAs in vitro, its role in rRNA extracts of cells. Depletion of Rpp2 protein in vivo causes processing is affected by proteins that associate with RNase P accumulation of precursor tRNAs with unprocessed introns in vivo (11–14). RNase P functions in the biosynthesis of and 5* and 3* termini, and leads to defects in the processing tRNAs (8) and appears to have a role in rRNA processing in of the 35S precursor rRNA. Rpp2-depleted cells are defective yeast (14, 24). Thus, processing of precursor tRNA and rRNA in processing of the 5.8S rRNA. Rpp2 immunoprecipitates may be coordinated by the activity of these two related cleave both yeast precursor tRNAs and precursor rRNAs enzymes (14). accurately at the expected sites and contain the Rpp1 protein We report here the cloning and functional characterization orthologue of the human scleroderma autoimmune antigen, of an essential (Rpp2, 15.8-kDa) subunit of S. cerevisiae Rpp30. These results demonstrate that Rpp2 is a protein nuclear RNase P. The gene for this protein, RPP2, was subunit of nuclear RNase P that is functionally conserved in identified by virtue of its homology with a human Rpp20, eukaryotes from yeast to humans. which copurifies with human RNase P (10, 25). The role of Rpp2 protein in processing of precursor tRNA and the 35S RNase P is a ubiquitous endoribonuclease that cleaves 59 precursor rRNA is similar to that of Rpp1, a previously terminal leader sequences of precursor tRNAs to generate 59 characterized yeast RNase P protein subunit (14). A condi- mature termini of tRNAs (1, 2). It is a ribonucleoprotein tional lethal yeast strain of Rpp2 revealed multiple defects in enzyme composed of RNA and protein subunits. Eubacterial processing of precursor tRNAs and precursor 35S rRNA. RNase P consists of a single catalytic RNA subunit (3) and a These results demonstrate that Rpp2 is a protein subunit of single, small, very basic protein cofactor ('14 kDa) that nuclear RNase P that is functionally conserved in eukaryotes increases the catalytic activity and substrate range of the from yeast to humans (10, 12, 14, 25). holoenzyme (4, 5). Archaeal and eukaryotic RNase P RNA have not been shown to be catalytic in vitro in the absence of MATERIALS AND METHODS protein subunits, despite the genetic, biochemical, and phylo- genetic evidence which showed that the RNA subunits of these Strains, Media, and General Procedures. S. cerevisiae strains enzymes are essential for activity and share significant struc- used in this study are VS200 (MATayMATa GAL1yGAL1 tural similarities to the eubacterial RNAs (6–9). The depen- ade2–101yade2–101 leu2–3, 112yleu2–3, 112 ura5–52yura5–52 dence of eukaryotic RNase P on protein components suggests his3-D200yhis3-D200 lys2–801ylys2–801 trp1-D63ytrp1-D63 that the protein subunits may be required for a direct role in RPP2yrpp2::HIS3), VS202A (MATa GAL1 ade2–101 leu2–3, the catalytic activity of the enzyme. However, the exact 112 ura5–52 his3-D200 lys2–801 trp1-D63 rpp2::HIS3 1 identity of all the eukaryotic protein subunits has not yet been pRS314FHRPP2(TRP1)), VS203 (MATa GAL1 ade2–101 determined. At least seven proteins are associated with highly leu2–3, 112 ura5–52 his3-D200 lys2–801 trp1-D63 rpp2::HIS3 1 purified RNase P from human cells: Rpp14, Rpp20, Rpp25, pRS314FHRPP2(TRP1) 1 pRS3163xRPP1(URA3)), VS211B Rpp29, Rpp30, Rpp38, and Rpp40 (10). Genetic and biochem- (MATa GAL1 ade2–101 leu2–3, 112 ura5–52 his3-D200 lys2– ical approaches in Saccharomyces cerevisiae identified four 801 trp1-D63 rpp2::HIS3 1 pRS314RPP2(TRP1)), VS214 proteins—Pop1, Pop3, Pop4 (homologue of Rpp29), and Rpp1 (MATayMATa GAL1yGAL1 ade2–101yade2–101 leu2–3, (homologue of Rpp30)—that associate with RNase P; these 112yleu2–3, 112 ura5–52yura5–52 his3-D200yhis3-D200 lys2– proteins also associate with a related enzyme called RNase 801ylys2–801 trp1-D63ytrp1-D63), VS301A (MATa GAL1 mitochondrial RNA processing (RNase MRP; refs. 11–14). ade2–101 leu2–3, 112 ura5–52 his3-D200 lys2–801 trp1-D63 Yeast nuclear RNase P and RNase MRP are related ribo- rpp2::HIS3 1pYCPGAL::rpp2 (URA3)), and VS301D (MATa nucleoprotein (RNP) enzymes essential for biosynthesis of GAL1 ade2–101 leu2–3, 112 ura5–52 his3-D200 lys2–801 trp1- tRNAs and rRNAs (15–18). The RNA component of RNase D63 RPP2 1pYCPGAL(URA3)). Genetic techniques and MRP has secondary structure similarity to RNase P RNA (19), preparations of standard media were performed according to and both enzymes appear to share protein subunits in vivo.It Abbreviations: Rpp2, RNase P protein 2; ptRNA, precursor tRNA; The publication costs of this article were defrayed in part by page charge prRNA, precursor rRNA, RNase MRP, RNase mitochondrial RNA processing; ITS, internal transcribed sequences. payment. This article must therefore be hereby marked ‘‘advertisement’’ in Data deposition: The sequence reported in this paper has been accordance with 18 U.S.C. §1734 solely to indicate this fact. deposited in the GenBank database (accession no. AF055991). © 1998 by The National Academy of Sciences 0027-8424y98y956716-6$2.00y0 ‡Towhomreprintrequestsshouldbeaddressed.e-mail:sidney.altman@ PNAS is available online at http:yywww.pnas.org. qm.yale.edu. 6716 Downloaded by guest on September 30, 2021 Biochemistry: Stolc et al. Proc. Natl. Acad. Sci. USA 95 (1998) 6717 established procedures (33). The identities of all constructs pRS314FHRPP2. To generate the GAL::RPP2 construct, the were verified by sequencing. Oligonucleotides used in this RPP2 coding sequence was subcloned from pRPP2SK as a study are: RPP2HIS5, 59-AACGAGTAACGAAACACCCA- 0.9-kb BstNI fragment into pYCPGAL(URA3) to generate TCTTTGAAAACTCTAACGCATAAGCCTCGTTCAGA- pYCPGAL::rpp2. ATGACACG-39; RPP2HIS3, 59-ATAGTATACAATAACA- Strain Construction. RPP2-disrupted diploid strain VS200 ATGAGCGCTAAAAGTATGACATATTAAACTCTTG- was transformed with pRS314FHRPP2 and sporulated. The GCCTCCTCTAG-39; RPP2FH, 59-TGCACCCAGGATGG- resulting spores were dissected, and spores that had a disrupted ACTACAAGGATGACGATGACAAGCATCATCATCAT- RPP2 gene but harbored pRS314FHRPP2 plasmid were viable CATCATGCTATGGCACTCAAAAAGAATACAC-39; (VS202A). The two haploids that depend on the plasmid RPP2J, 59-GCCATCCTGGGTGCAG-39; RPP2R, 59-TCCC- pRS314FHRPP2 grew at rates identical to the wild-type hap- CGCGGGGACGGCCGCTTCATCAATTCTGTTTACCC- loids (VS211B). VS202A also was transformed with TTG-39; RPP2T, 59-CGGAATTCGTTTATCAAGTGGAT- pRS316–3xmyc::RPP1 (14) and grown in media lacking uracil, ATGC-39; oligonucleotide a is oligonucleotide no. 5 in ref. 14; to generate VS203. VS200 was transformed with oligonucleotide b, 59-GGCCAGCAATTTCAAGT-39; pYCPGAL::rpp2 sporulated, and germinated on medium con- SNR190, 59-GGCTCAGATCTGCATGTGTTG-39. taining galactose. Viable spores were obtained from several Gene Disruption. A genomic clone encoding the RPP2 locus independent tetrads. Haploids with a disrupted RPP2 allele was identified in the S. cerevisiae genome database (SGD) and that depend on pYCPGAL::rpp2 (VS301A) grew on galactose- obtained from the American Tissue Culture Collection containing plates but not on glucose-containing plates (data (ATCC) as a cosmid (ATCC 71051), which was sequenced not shown). previously. A 2.2-kb AflII fragment that encodes RPP2 was Other Methods. Immunoprecipitations, RNase P and subcloned into pBluescript (SK) vector to generate pRPP2SK. RNase MRP enzymatic assays, and RNA extraction and Using oligonucleotides RPP2HIS5 and RPP2HIS3, the HIS3 analysis were performed as described; immunoprecipitates gene was amplified by PCR from plasmid pRS313(HIS3). The were washed in IP150 buffer (14). Primer extension analysis of resulting 1.2-kb fragment was gel-purified and then was inte- the cleavage of internal transcribed sequences 141 (ITS141) grated at the RPP2 genomic locus in the diploid yeast strain prRNA substrate at the A3 site was performed by using VS214 to generate VS200. The correct replacement of one oligonucleotide 6 (14), according to manufacturer’s procedure allele was verified by PCR digest analysis. Oligonucleotides for reverse transcriptase (Promega). RPP2R and RPP2T were used to amplify genomic DNA isolated from VS200, and the resulting (1.6-kb) fragment RESULTS encoding HIS3 and flanking sequences of RPP2 was mapped with restriction enzymes. An Essential Yeast Gene Encodes a Homologue of Human Construction of Plasmids. A plasmid encoding the FLAG- Rpp20, a Protein Subunit of Nuclear RNase P. We identified HIS epitope-tagged RPP2, pRS314FHRPP2(TRP1), was gen- the RPP2 gene by searching the SGD (26) with the protein erated by PCR using oligonucleotides RPP2FH, RPP2J, sequence of the human Rpp20 protein, which copurifies with RPP2R, RPP2T, and pRPP2SK as template. The PCR frag- human nuclear RNase P (10, 25). We used a computational ment was subcloned into pRS314(TRP1) plasmid to generate sequence search algorithm (BLASTP) (27) to identify a previ- FIG. 1. Yeast Rpp2 is homologous to the human Rpp20 protein.