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• W2134714863 abstract "The RNase MRP and RNase P particles both function as endoribonucleases. RNase MRP has been implicated in the processing of precursor-rRNA, whereas RNase P has been shown to function in the processing of pre-tRNA. Both ribonucleoprotein particles have an RNA component that can be folded into a similar secondary structure and share several protein components. We have identified human, rat, mouse, cow, and Drosophila homologues of the Pop5p protein subunit of the yeast RNase MRP and RNase P complexes. The human Pop5 cDNA encodes a protein of 163 amino acids with a predicted molecular mass of 18.8 kDa. Polyclonal antibodies raised against recombinant hPop5 identified a 19-kDa polypeptide in HeLa cells and showed that hPop5 is associated with both RNase MRP and RNase P. Using affinity-purified anti-hPop5 antibodies, we demonstrated that the endogenous hPop5 protein is localized in the nucleus and accumulates in the nucleolus, which is consistent with its association with RNase MRP and RNase P. Catalytically active RNase P was partially purified from HeLa cells, and hPop5 was shown to be associated with it. Finally, the evolutionarily conserved acidic C-terminal tail of hPop5 appeared to be required neither for complex formation nor for RNase P activity. The RNase MRP and RNase P particles both function as endoribonucleases. RNase MRP has been implicated in the processing of precursor-rRNA, whereas RNase P has been shown to function in the processing of pre-tRNA. Both ribonucleoprotein particles have an RNA component that can be folded into a similar secondary structure and share several protein components. We have identified human, rat, mouse, cow, and Drosophila homologues of the Pop5p protein subunit of the yeast RNase MRP and RNase P complexes. The human Pop5 cDNA encodes a protein of 163 amino acids with a predicted molecular mass of 18.8 kDa. Polyclonal antibodies raised against recombinant hPop5 identified a 19-kDa polypeptide in HeLa cells and showed that hPop5 is associated with both RNase MRP and RNase P. Using affinity-purified anti-hPop5 antibodies, we demonstrated that the endogenous hPop5 protein is localized in the nucleus and accumulates in the nucleolus, which is consistent with its association with RNase MRP and RNase P. Catalytically active RNase P was partially purified from HeLa cells, and hPop5 was shown to be associated with it. Finally, the evolutionarily conserved acidic C-terminal tail of hPop5 appeared to be required neither for complex formation nor for RNase P activity. expressed sequence tag polymerase chain reaction glutathione S-transferase open reading frame vesicular stomatitis virus The RNase MRP/RNase P ribonucleoprotein particles form one of the three families of small nucleolar ribonucleoprotein complexes that are involved in the processing of precursor rRNA to mature 5.8, 18, and 25/28 S rRNA (reviewed in Ref. 1Tollervey D. Kiss T. Curr. Opin. Cell Biol. 1997; 9: 337-342Crossref PubMed Scopus (377) Google Scholar). The RNase MRP ribonucleoprotein particle has originally been identified by virtue of its capacity to cleave a mitochondrial RNA in vitro to generate RNA primers for mitochondrial DNA replication (2Chang D.D. Clayton D.A. EMBO J. 1987; 6: 409-417Crossref PubMed Scopus (195) Google Scholar). However, most of the RNase MRP complex has been shown to reside in the nucleolus (3Chang D.D. Clayton D.A. Science. 1987; 235: 1178-1184Crossref PubMed Scopus (221) Google Scholar,4Reimer G. Raska I. Scheer U. Tan E.M. Exp. Cell Res. 1988; 176: 117-128Crossref PubMed Scopus (88) Google Scholar). There, it functions in the formation of the short form of the 5.8 S rRNA (5.8 S(S)) by cleaving at site A3 in the internal transcribed spacer 1 (ITS1) of precursor rRNA (5Chu S. Archer R.H. Zengel J.M. Lindahl L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 659-663Crossref PubMed Scopus (176) Google Scholar, 6Lygerou Z. Allmang C. Tollervey D. Seraphin B. Science. 1996; 272: 268-270Crossref PubMed Scopus (214) Google Scholar, 7Lygerou Z. Mitchell P. Petfalski E. Seraphin B. Tollervey D. Genes Dev. 1994; 8: 1423-1433Crossref PubMed Scopus (179) Google Scholar, 8Schmitt M.E. Clayton D.A. Mol. Cell. Biol. 1993; 13: 7935-7941Crossref PubMed Scopus (230) Google Scholar). In many different aspects, the RNase MRP complex is related to the RNase P complex, a ribonucleoprotein complex required for the removal of the 5′ end of the precursor tRNAs. Both function as site-specific endonucleases, contain an RNA component that has been proposed to adopt a similar cage-shaped structure, share several protein subunits, and are predominantly localized in the nucleolus (reviewed in Ref. 9van Eenennaam H. Jarrous N. van Venrooij W.J. Pruijn G.J.M. IUBMB Life. 2000; 49: 265-272Crossref PubMed Scopus (49) Google Scholar). The biological importance of the RNase MRP function is substantiated by the recent observations that mutations in the gene encoding the RNA component of the human RNase MRP complex cause an autosomal recessive disease called cartilage-hair hypoplasia (10Ridanpaa M. van Eenennaam H. Pelin K. Chadwick R. Johnson C. Yuan B. vanVenrooij W. Pruijn G. Salmela R. Rockas S. Makitie O. Kaitila I. de la Chapelle A. Cell. 2001; 104: 195-203Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). In addition, RNase MRP and RNase P play a role in certain autoimmune diseases. Protein components of the RNase MRP and RNase P complexes are targeted by autoantibodies in systemic lupus erythematosus and scleroderma (11Gold H.A. Topper J.N. Clayton D.A. Craft J. Science. 1989; 245: 1377-1380Crossref PubMed Scopus (109) Google Scholar, 12Gold H.A. Craft J. Hardin J.A. Bartkiewicz M. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5483-5487Crossref PubMed Scopus (62) Google Scholar, 13Hashimoto C. Steitz J.A. J. Biol. Chem. 1983; 258: 1379-1382Abstract Full Text PDF PubMed Google Scholar, 14Reddy R. Tan E.M. Henning D. Nohga K. Busch H. J. Biol. Chem. 1983; 258: 1383-1386Abstract Full Text PDF PubMed Google Scholar, 15Hardin J.A. Rahn D.R. Shen C. Lerner M.R. Wolin S.L. Rosa M.D. Steitz J.A. J. Clin. Invest. 1982; 70: 141-147Crossref PubMed Scopus (80) Google Scholar). The first identified protein subunit of the human RNase MRP and RNase P complexes is the hPop1 protein, which is a homologue of the yeast Pop1p protein (16Lygerou Z. Pluk H. van Venrooij W.J. Seraphin B. EMBO J. 1996; 15: 5936-5948Crossref PubMed Scopus (82) Google Scholar). Most of the currently known human protein subunits of the RNase MRP and RNase P complexes have been identified via purification of the RNase P complex from HeLa cells: Rpp14, Rpp20, Rpp29/hPop4, Rpp30, Rpp38, and Rpp40 (17Eder P.S. Kekuda R. Stolc V. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1101-1106Crossref PubMed Scopus (98) Google Scholar, 18Jarrous N. Eder P.S. Wesolowski D. Altman S. RNA. 1999; 5: 153-157Crossref PubMed Scopus (48) Google Scholar, 19van Eenennaam H. Pruijn G.J. van Venrooij W.J. Nucleic Acids Res. 1999; 27: 2465-2472Crossref PubMed Scopus (39) Google Scholar, 20Jarrous N. Eder P.S. Guerrier Takada C. Hoog C. Altman S. RNA. 1998; 4: 407-417PubMed Google Scholar). In yeast 10 proteins have been found to be associated with RNase MRP or RNase P or with both complexes (reviewed in Ref. 9van Eenennaam H. Jarrous N. van Venrooij W.J. Pruijn G.J.M. IUBMB Life. 2000; 49: 265-272Crossref PubMed Scopus (49) Google Scholar). Snm1p has been reported to be associated specifically with the RNase MRP complex, whereas the Rpr2p protein is only bound to the RNase P complex (21Schmitt M.E. Clayton D.A. Genes Dev. 1994; 8: 2617-2628Crossref PubMed Scopus (81) Google Scholar, 22Chamberlain J.R. Lee Y. Lane W.S. Engelke D.R. Genes Dev. 1998; 12: 1678-1690Crossref PubMed Scopus (206) Google Scholar). Other subunits shared by RNase MRP and RNase P in yeast are: Pop1p, Pop7p/Rpp2p, Pop4p, Rpp1p, Pop3p, Pop5p, Pop6p, and Pop8p (7Lygerou Z. Mitchell P. Petfalski E. Seraphin B. Tollervey D. Genes Dev. 1994; 8: 1423-1433Crossref PubMed Scopus (179) Google Scholar, 22Chamberlain J.R. Lee Y. Lane W.S. Engelke D.R. Genes Dev. 1998; 12: 1678-1690Crossref PubMed Scopus (206) Google Scholar, 23Stolc V. Altman S. Genes Dev. 1997; 11: 2926-2937Crossref PubMed Scopus (46) Google Scholar, 24Stolc V. Katz A. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6716-6721Crossref PubMed Scopus (34) Google Scholar, 25Dichtl B. Tollervey D. EMBO J. 1997; 16: 417-429Crossref PubMed Scopus (75) Google Scholar, 26Chu S. Zengel J.M. Lindahl L. RNA. 1997; 3: 382-391PubMed Google Scholar). Although homologues for several of the yeast proteins are found in human cells (Pop1p, Pop4p, Pop7p/Rpp2p, Rpp1p), until now no homologues have been identified for Snm1p, Pop3p, Pop5p, Pop6p, Pop8p, and Rpr2p (reviewed in Ref. 9van Eenennaam H. Jarrous N. van Venrooij W.J. Pruijn G.J.M. IUBMB Life. 2000; 49: 265-272Crossref PubMed Scopus (49) Google Scholar). In this report we describe the identification, cDNA cloning, and characterization of the human Pop5 protein, which, like all human protein subunits identified so far, is associated with both RNase MRP and RNase P RNAs. Data base searches were performed using the BLASTN 2.0.10 program (27Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59759) Google Scholar). The accession numbers of the human expressed sequence tags (ESTs)1 are AF117232, XM-012126, NM-015918, and AF070660. The accession numbers for the overlapping mouse ESTs are BF121181 and BF583365, for rat ESTs BF567090and AW918527, for cow BF075658, and for Drosophila melanogaster AAF49372. The following oligonucleotides (based on the human EST sequence) were used to isolate the open reading frame of hPop5 by a PCR-based approach: pop51 (5′-GCG-GAT-CCC-TCG-AGA-TGG-TGC-GGT-TCA-AGC-ACA-GG-3′) and pop52 (5′-GCG-GAT-CCC-CCG-GGT-CAT-CTA-GAC-TCC-ATT-GCT-TCT-GCA-GCC-TCC-TC-3′). The polymerase chain reaction was performed with 200 ng of denatured DNA from λgt11 human placenta (CLONTECH) and teratocarcinoma cDNA libraries as starting material (28Sillekens P.T. Habets W.J. Beijer R.P. van Venrooij W.J. EMBO J. 1987; 6: 3841-3848Crossref PubMed Scopus (146) Google Scholar). The amplified fragments were ligated in the PCR-II-TOPO vector (Invitrogen) and sequenced using the dideoxynucleotide chain termination method. To raise a polyclonal anti-hPop5 antiserum, the hPop5 protein was expressed as a fusion protein with glutathione S-transferase (GST) in E. coli and purified by glutathione affinity chromatography (29Frangioni J.V. Neel B.G. Anal. Biochem. 1993; 210: 179-187Crossref PubMed Scopus (831) Google Scholar). Rabbits were immunized with this material following standard procedures (30Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar). For each injection 200 µg of GST-hPop5 was used. To obtain anti-hPop5 and anti-GST antibodies, two affinity columns were prepared by immobilizing either 1.25 mg of GST-hPop5 or 4.8 mg of GST protein to 1.5 g of CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Rabbit serum raised against GST-hPop5 was first depleted of anti-GST activity by chromatography on the GST affinity column equilibrated with Buffer A (350 mm NaCl, 0.05% Nonidet P-40, 1× PBS). Anti-GST antibodies were eluted from the column using Buffer B (0.1m glycine-HCl, pH 2.5, 0.5 m NaCl, 0.05% Nonidet P-40). This procedure was repeated until no detectable anti-GST activity could be measured in the flow-through using enzyme-linked immunosorbent assay methods. Subsequently, the flow-through was loaded on the GST-hPop5 column in Buffer A. After washing with Buffer A, the anti-hPop5 antibodies were eluted with Buffer B. For Western blot analysis, the rabbit anti-hPop5 and pre-immune sera were used in a 500-fold dilution, whereas affinity-purified anti-GST and anti-hPop5 antibodies were used in a 100-fold dilution. Detection was performed using horseradish peroxidase-conjugated goat anti-rabbit IgG (Dako Immunoglobulins) as secondary antibody and visualization by chemiluminescence. Extracts of HEp-2 cells were prepared by resuspending cell pellets in buffer A (25 mm Tris-HCl, pH 7.5, 100 mm KCl, 1 mm dithioerythritol, 2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 0.05% Nonidet P-40) and lysis by sonication using a Branson microtip (three times for 20 s each). Insoluble material was removed by centrifugation (12,000 ×g, 15 min) and supernatants were used directly for immunoprecipitations. A monoclonal anti-vesicular stomatitis virus (VSV) tag antibody (Roche), an anti-55K (31Pluk H. Soffner J. Luhrmann R. van Venrooij W.J. Mol. Cell. Biol. 1998; 18: 488-498Crossref PubMed Scopus (41) Google Scholar) antiserum, a patient anti-Th/To serum, the rabbit anti-hPop5 antiserum, an anti-hPop4 antiserum (19van Eenennaam H. Pruijn G.J. van Venrooij W.J. Nucleic Acids Res. 1999; 27: 2465-2472Crossref PubMed Scopus (39) Google Scholar), and pre-immune serum from the rabbit immunized with hPop5 protein were coupled to protein A-agarose beads (Biozym) in IPP500 (500 mm NaCl, 10 mm Tris-HCl, pH 8.0, 0.05% Nonidet P-40) by incubation for 1 h at room temperature. Beads were washed twice with IPP500 and once with IPP150 (150 mm NaCl, 10 mm Tris-HCl, pH 8.0, 0.05% Nonidet P-40). For each immunoprecipitation a cell extract was incubated with the antibody-coupled beads for 2 h at 4 °C. Subsequently, beads were washed three times with IPP150. To analyze co-precipitating RNAs, the RNA was isolated by phenol-chloroform extraction and ethanol precipitation. RNAs were resolved on a denaturing polyacrylamide gel and blotted to a Hybond-N membrane (Amersham Pharmacia Biotech). Northern blot hybridizations with riboprobes specific for human RNase P, RNase MRP, and U3 RNAs were performed as described previously (32Verheijen R. Wiik A. De Jong B.A. Hoier Madsen M. Ullman S. Halberg P. van Venrooij W.J. J. Immunol. Methods. 1994; 169: 173-182Crossref PubMed Scopus (22) Google Scholar). Indirect immunofluorescence assays with affinity-purified rabbit anti-hPop5 antibodies and affinity-purified rabbit anti-GST antibodies were performed on HEp-2 cells. Fixed cells were incubated with affinity-purified antibodies (diluted 1:20 in PBS) for 1 h at room temperature, washed with PBS, and subsequently incubated with Alexa Fluor® 488 goat anti-rabbit IgG conjugate (Molecular Probes, diluted 1:75 in PBS) for 1 h at room temperature. Bound antibodies were visualized by epifluorescence microscopy. Purification of the human RNase P enzyme was performed as described previously (17Eder P.S. Kekuda R. Stolc V. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1101-1106Crossref PubMed Scopus (98) Google Scholar, 33Bartkiewicz M. Gold H. Altman S. Genes Dev. 1989; 3: 488-499Crossref PubMed Scopus (138) Google Scholar). HeLa S3 cells (National Cell Culture Center, Minneapolis, MN) were pelleted, disrupted, and the cell lysate was centrifuged at 10,000 × g, followed by another centrifugation at 100,000 g. The extract was loaded on a DEAE-Sepharose column, and bound material was eluted with a linear 100–500 mm KCl gradient. Fractions containing RNase P activity were pooled, concentrated, and further fractionated by centrifugation in a 15–30% glycerol gradient. To assay for RNase P activity in the immunoprecipitates and column fractions, an internally32P-labeled pre-tRNA substrate (Schizosaccharomyces pombe tRNASer SupS1), was transcribed in vitro and gel-purified. This 110-nucleotide-long substrate contains a 5′ end extension of 28 nucleotides in comparison with the mature tRNA. The immunoprecipitates or fractions were incubated with equal amounts of substrate in assay buffer (20 mm Tris-HCl, pH 8.0, 10 mm MgCl2, 1 mmdithioerythritol, 50 mm KCl, 50 µg/ml bovine serum albumin, 60 units/ml RNasin) for 10 min at 37 °C. Reaction products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. VSV G epitope (34Kreis T.E. EMBO J. 1986; 5: 931-941Crossref PubMed Scopus (283) Google Scholar) (VSV-G)-tagged (hereafter referred to as VSV-tagged) cDNAs were constructed as follows. 55k-VSV cDNA constructs (31Pluk H. Soffner J. Luhrmann R. van Venrooij W.J. Mol. Cell. Biol. 1998; 18: 488-498Crossref PubMed Scopus (41) Google Scholar), containing either aXhoI or a XbaI site between the 55-kDa open reading frame (ORF) and the VSV tag sequence (positioned at the N- and C-terminal sides, respectively) were cleaved with XhoI andXbaI releasing the 55-kDa cDNA from this plasmid. The VSV-tagged constructs of hPop5 were constructed by isolation of the hPop5 ORF from the hPop5/PCR-II-TOPO construct byXhoI/XbaI digestion and ligation into theXhoI/XbaI-digested 55k-VSV constructs. A construct expressing deletion mutant hPop5 Δ(145–163) was generated by a PCR-based approach. Besides the deletion introduced by the PCR strategy, an XhoI site was introduced at the 5′ end of the translational start codon, whereas an XbaI site was introduced at the 3′ end of the coding sequence. The transfection construct was generated by ligating the XhoI/XbaI fragment into the XhoI/XbaI-digested 55k-VSV constructs. The integrity of the resulting construct was confirmed by DNA sequencing. The pCI-neo vector (Promega), which has been used to prepare 55k-VSV previously, was used as a control in the transfection experiments. HEp-2 monolayer cells were grown to 70% confluence by standard tissue culture techniques in either culture flasks or on coverslips. HEp-2 cells were transfected with the VSV-tagged constructs (3–4 µg) using LipofectAMINE 2000 (Life Technologies, Inc.) according to the manufacturer's instructions. After overnight growth in flasks, cells were harvested, washed once with PBS, and used to prepare extracts for immunoprecipitation assays. Purification of the yeast RNase P holoenzyme has resulted in the identification of nine protein subunits (22Chamberlain J.R. Lee Y. Lane W.S. Engelke D.R. Genes Dev. 1998; 12: 1678-1690Crossref PubMed Scopus (206) Google Scholar). Although some of these were shown to have homologues in humans, no human homologues have been reported for Snm1p, Pop3p, Pop5p, Pop6p, Pop8p, and Rpr2p. We have searched protein and “translated nucleic acid” sequence data bases to identify sequences that might represent human homologues of these yeast protein subunits. Four virtually identical nucleic acid entries were found that encode a putative human homologue of the yeast Pop5p protein (hereafter designated yPop5). The latter polypeptide consists of 173 amino acids and has a predicted molecular mass of 19.6 kDa. The corresponding human sequence (data base entry AF117232) is 785 nucleotides long and contains an ORF encoding a polypeptide of 162 amino acids with a predicted molecular mass of 18.7 kDa. For reasons documented below, this protein will be referred to as hPop5. Besides the human ESTs retrieved from the sequence data bases, two mouse ESTs, two rat ESTs, one cow EST, and one D. melanogaster entry that encode putative homologues of the yeast Pop5p protein were identified. These entries contain ORFs of 169, 169, 170, and 145 amino acids, respectively. To obtain a cDNA encoding the putative human Pop5 protein, two oligonucleotides were designed based on the identified human EST sequence. These oligonucleotides were used as PCR primers to isolate hPop5 cDNAs from human teratocarcinoma and placenta cDNA libraries. Sequence analysis of several clones obtained by this procedure revealed that cDNAs derived from both sources were identical, thereby ruling out the introduction of PCR artifacts. Two minor differences were found in all sequenced cDNAs in comparison with the corresponding EST described above (AF117232). Nucleotides 154–162 in the cloned cDNA sequence (numbering according to data base accession no. AJ306296) are 5′-GCA GCCGCC-3′, whereas the EST contains 5′-GCA CCC-3′ at this position, resulting in the replacement of a Pro codon by two Ala codons in the cloned cDNA in comparison with the EST. Furthermore, a single nucleotide substitution was detected at position 461, where a T residue was found rather than a C residue, which is observed at this position in the human EST. To determine whether the first ATG present in the human EST represents the translational start codon, we synthesized cDNA from several sources of mRNA. DNA sequencing of the resulting clones revealed that no additional sequence information was obtained (data not shown). The cloned cDNA encodes a protein of 163 amino acids, with a predicted molecular mass of 18.8 kDa and a predicted pI of 7.9. In Fig. 1an alignment of the amino acid sequences derived from the human, cow, rat, mouse,Drosophila, and yeast cDNAs/ESTs is shown. This illustrates the high level of homology between the mammalian Pop5 polypeptides (88–91% identity, 93–95% similarity), whereas the homology between the human, Drosophila, and yeast proteins is much lower (23–27% identity and 40–43% similarity). The conservation of the yeast and human Pop5 amino acid sequences is most extensive in the N terminus. Except for the acidic C terminus, the primary sequence of hPop5 does not reveal established sequence motifs. The acidic nature of the C terminus is conserved from human to yeast (in hPop5, 9 out of 15 residues are acidic), although it appears to be absent in the Drosophila sequence. To determine whether the hPop5 protein is associated with the human RNase MRP and RNase P ribonucleoprotein particles, a polyclonal antibody was raised against recombinant hPop5. The hPop5 protein was expressed as a fusion protein with GST in E. coli. The recombinant GST-hPop5 fusion protein was purified using glutathione-Sepharose 4B beads (Fig. 2,lane 2) and used for immunization of rabbits. Western blot analysis showed that the resulting rabbit serum is reactive with the GST-hPop5 protein (lane 4), whereas the pre-immune serum is not (lane 3). Because the rabbit serum failed to recognize the hPop5 protein in HeLa extracts (data not shown), anti-hPop5 antibodies were purified by affinity selection with recombinant GST-hPop5 from the rabbit serum. Prior to the GST-hPop5 selection, the rabbit serum was depleted of anti-GST activity by performing affinity selection with recombinant GST. Western blot analysis of HeLa cell extracts with the affinity-purified antibodies against hPop5 revealed a single protein with an estimated size of 19 kDa (lane 6), whereas the affinity-purified antibodies against GST failed to detect any protein in HeLa extracts (lane 5). To investigate whether the hPop5 protein is associated with the RNase MRP and RNase P complexes, immunoprecipitations with the anti-hPop5 serum using total HeLa cell extract were performed. The co-precipitating RNAs were isolated and analyzed by Northern blot hybridization using riboprobes specific for RNase MRP, RNase P, and U3 RNA. As depicted in Fig. 3(lane 6), the anti-hPop5 antiserum efficiently immunoprecipitated both RNase MRP and RNase P RNAs, whereas no detectable precipitation of U3 RNA was observed. The specificity of this assay was demonstrated by the observation that pre-immune serum did not precipitate any of the RNAs analyzed (lane 5) and that anti-55K antibodies specifically precipitated U3 RNA (lane 3) in accordance with previous observations (31Pluk H. Soffner J. Luhrmann R. van Venrooij W.J. Mol. Cell. Biol. 1998; 18: 488-498Crossref PubMed Scopus (41) Google Scholar). In addition, the RNase MRP and RNase P RNAs were co-immunoprecipitated by a patient anti-Th/To antiserum and by anti-hPop4 antibodies (lanes 2 and 4). These results indicate that the 19-kDa hPop5 protein is associated with both RNase MRP and RNase P RNAs in HeLa cells. To investigate the subcellular localization of the hPop5 protein, the affinity-purified antibodies directed against GST-hPop5 and GST were used for immunolocalization experiments in HEp-2 cells. The affinity-purified anti-hPop5 antibodies strongly stained the nucleoli and showed, in addition, a fine-speckled cytoplasmic staining (Fig.4, panel A). Using affinity-purified anti-GST antibodies, a similar fine-speckled cytoplasmic staining pattern, but no nucleolar staining, was observed (panel C), strongly suggesting that the cytoplasmic staining is caused by anti-GST reactivity. Although these results do not completely rule out a cytoplasmic localization of the hPop5 protein, they clearly show that the hPop5 protein accumulates in the nucleoli, consistent with its association with RNase MRP and RNase P. To obtain further evidence that the hPop5 protein is part of the complete RNase P complex, we tested whether hPop5 is associated with catalytically active RNase P. First, we partially purified the active RNase P complex from a HeLa cell extract by DEAE-Sepharose chromatography and glycerol gradient centrifugation. The fractions of the glycerol gradient were tested for RNase P activity by monitoring its ability to specifically cleave an internally labeled precursor tRNA substrate to tRNA and its 5′-leader. Products of this reaction were separated on a denaturing polyacrylamide gel and visualized by autoradiography. In agreement with previous observations (17,33), the RNase P activity was only detected in relatively fast sedimenting fractions (Fig. 5A). To study the presence of the hPop5 protein in the active RNase P fractions of the glycerol gradient, these fractions were analyzed by immunoblotting. As shown in the lower part of Fig. 5 A, hPop5 (bottom panel), hPop4 (middle panel), and Rpp38 (top panel) co-fractionated with the RNase P activity, suggesting that hPop5 indeed is associated with catalytically active RNase P. To confirm this, immunoprecipitations were performed on RNase P containing glycerol gradient fractions using affinity-purified anti-hPop5 and anti-GST antibodies and the immunoprecipitates were assayed for RNase P activity. As shown in Fig. 5 B (lanes 2and 3), the immunoprecipitate obtained with the affinity-purified anti-hPop5 antibodies was able to cleave pre-tRNA into mature tRNA and its 5′-leader, whereas the immunoprecipitate of the affinity-purified anti-GST antibodies was not. The RNase P activity in the anti-hPop5 immunoprecipitate was indistinguishable from that obtained with anti-hPop4 antibodies, which functioned as a positive control (lane 1). These results indicate that the hPop5 protein is part of the catalytically active RNase P complex. Although these data do not show that the hPop5 protein is associated with catalytically active RNase MRP as well, the strong evolutionary relationship between the RNase P and RNase MRP complexes suggests that hPop5 is also associated with RNase MRP. As described above, the only amino acid sequence element that could be detected in the primary structure of hPop5 is its acidic C terminus. Therefore, we investigated whether this sequence element is required for complex association. A VSV tag sequence (34Kreis T.E. EMBO J. 1986; 5: 931-941Crossref PubMed Scopus (283) Google Scholar) was fused to the 3′ end of the hPop5 ORF and to the 3′ end of a truncated cDNA lacking the sequence corresponding to the acidic C terminus (designated hPop5-VSV and hPop5 Δ(145–163), respectively). HEp-2 cells were transfected with these constructs or with the corresponding “empty” vector as a negative control. After culturing the cells for 16-h extracts were prepared and used for immunoprecipitation with anti-VSV tag antibodies. Co-precipitating RNAs were isolated and analyzed via Northern blot hybridization using riboprobes for RNase MRP and RNase P RNA. As is shown in Fig.6A (lanes 1 and 2), RNase MRP and RNase P RNA were both immunoprecipitated from a cell extract containing the hPop5-VSV protein, but not from an extract from cells transfected with the empty vector. This result confirms that hPop5 associates with the RNase MRP and RNase P RNAs in human cells and demonstrates that this approach is suitable to analyze hPop5 mutants. As depicted in lane 3, the hPop5 Δ(145–163) mutant was able to associate with both RNase MRP and RNase P RNA, indicating that the evolutionarily conserved acidic C terminus is not required for association with these ribonucleoproteins. To investigate whether this element plays a role in the endoribonuclease activity of RNase P, HEp-2 cells were transfected with the VSV-tagged hPop5 and hPop5 Δ(145–163) constructs and immunoprecipitations were performed on extracts prepared from these cells. A 32P-labeled precursor tRNA was incubated at 37 °C with the immunoprecipitates, and the resulting products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. As shown in Fig. 6 B (lanes 1 and 2), complexes associated with both hPop5 and the mutant lacking the acidic C terminus displayed RNase P activity, whereas a control immunoprecipitate did not. Thus, the most C-terminal 19 amino acids of hPop5 are required neither for complex association nor for RNase P activity. In this report we describe the cloning of a novel human RNase MRP/RNase P protein subunit. Based upon its homology with the yeast Pop5p protein, we have designated this protein hPop5. We showed that the human protein, like the yeast Pop5p protein, is associated with both RNase MRP and RNase P ribonucleoprotein particles and that the latter particle is catalytically active in vitro. Besides the human homologue of Pop5p, our data base searches identified two ESTs (accession nos.AI751395 and AA534933) encoding a putative homologue of the RNase P-specific protein subunit: Rpr2p (9van Eenennaam H. Jarrous N. van Venrooij W.J. Pruijn G.J.M. IUBMB Life. 2000; 49: 265-272Crossref PubMed Scopus (49) Google Scholar). No homologues were identified for the Snm1p, Pop3p, Pop6p, and Pop8p protein, suggesting that only 6 of the 10 yeast RNase MRP/RNase P protein subunits have human counterparts. A high degree of sequence conservation was observed among the mammalian Pop5 proteins, whereas only a moderate level of conservation exists between human, yeast, and DrosophilaPop5 (23–27% identity). A similar level of sequence conservation has been reported for other RNase MRP and RNase P protein subunits: hPop1/Pop1p (22% identity), Rpp30/Rpp1p (23% identity), Rpp20/Rpp2p (14% identity), and hPop4/Pop4p (29% identity) (16Lygerou Z. Pluk H. van Venrooij W.J. Seraphin B. EMBO J. 1996; 15: 5936-5948Crossref PubMed Scopus (82) Google Scholar, 17Eder P.S. Kekuda R. Stolc V. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1101-1106Crossref PubMed Scopus (98) Google Scholar, 18Jarrous N. Eder P.S. Wesolowski D. Altman S. RNA. 1999; 5: 153-157Crossref PubMed Scopus (48) Google Scholar, 19van Eenennaam H. Pruijn G.J. van Venrooij W.J. Nucleic Acids Res. 1999; 27: 2465-2472Crossref PubMed Scopus (39) Google Scholar, 20Jarrous N. Eder P.S. Guerrier Takada C. Hoog C. Altman S. RNA. 1998; 4: 407-417PubMed Google Scholar). Recently, the analysis of the genomes of archae bacteria allowed the detection of hPop5 and Rpp30 counterparts also in these organisms (35Koonin E.V. Wolf Y.I. Aravind L. Genome Res. 2001; 11: 240-252Crossref PubMed Scopus (198) Google Scholar) and in addition showed that the secondary structures of Pop5 and Rpp14 are related. A similar conservation from humans to archaea has been suggested for the Rpp29/hPop4 and Rpp21 subunits (36Altman S. Gopalan V. Vioque A. RNA. 2000; 6: 1689-1694Crossref PubMed Scopus (9) Google Scholar). A comparison of the secondary structures of RNase MRP RNA and RNase P RNA from various organisms has led to the hypothesis that RNase MRP evolved from RNase P in an early eukaryote (37Collins L.J. Moulton V. Penny D. J. Mol. Evol. 2000; 51: 194-204Crossref PubMed Scopus (53) Google Scholar). The association of the four highly conserved proteins with MRP RNA would be explained by the hypothesis that proteins associated with RNase P RNA have the capacity to bind MRP RNA as well. We showed that the hPop5 protein is localized in the nucleus and accumulates in the nucleolus (Fig. 4). Previously, it has been proposed that nucleolar entry is a two-step mechanism (38Girard J.P. Bagni C. Caizergues Ferrer M. Amalric F. Lapeyre B. J. Biol. Chem. 1994; 269: 18499-18506Abstract Full Text PDF PubMed Google Scholar, 39Schmidt Zachmann M.S. Nigg E.A. J. Cell Sci. 1993; 105: 799-806Crossref PubMed Google Scholar, 40Yan C. Melese T. J. Cell Biol. 1993; 123: 1081-1091Crossref PubMed Scopus (58) Google Scholar). First, a nucleolar protein is transported to the nucleus and subsequently it accumulates in the nucleolus. Recently, we demonstrated that clusters of basic amino acids are important for the localization of protein subunits of RNase MRP and RNase P in the nucleolus. 2H. van Eenennaam, A. van der Heijden, R. J. R. J. Janssen, W. J. van Venrooij, and G. J. M. Pruijn, submitted for publication. The absence of a nuclear localization signal as well as clusters of basic residues in the hPop5 amino acid sequence suggests that hPop5 is targeted to the nucleus/nucleolus in a different manner. The hPop5 protein might be transported to the nucleus and nucleolus by a piggyback mechanism. In that case hPop5 binds in the cytoplasm to another (RNase MRP/RNase P) protein, which carries the hPop5 protein to the nucleus and subsequently to the nucleolus. Transport of the hPop5 protein from the nucleoplasm to the nucleolus might also be dependent on its association with the (partially assembled) RNase MRP/RNase P ribonucleoprotein complexes. A similar situation has been proposed for the Rpp14 protein (41Jarrous N. Wolenski J.S. Wesolowski D. Lee C. Altman S. J. Cell Biol. 1999; 146: 559-571Crossref PubMed Scopus (80) Google Scholar). The results in Figs. 3 and 6 demonstrate that the hPop5 protein subunit, like its yeast counterpart, is associated with both RNase MRP and RNase P. The association of protein subunits with both RNase MRP and RNase P has been previously reported for hPop1, Rpp30, Rpp38, and hPop4/Rpp29 (reviewed in Ref. 9van Eenennaam H. Jarrous N. van Venrooij W.J. Pruijn G.J.M. IUBMB Life. 2000; 49: 265-272Crossref PubMed Scopus (49) Google Scholar) and has been observed for the Rpp20 protein subunit as well. 3H. van Eenennaam, D. Lugtenberg, J. H. P. Vogelzangs, W. J. van Venrooij, and G. J. M. Pruijn, unpublished observations. The sharing of protein subunits by these endoribonuclease complexes might be explained at least in part by the conservation of the secondary structure of their RNA components and the suggestion that the RNase MRP RNA has evolved from the RNase P RNA in early eukaryotes (see above). In yeast two protein subunits have been described to be specifically associated with either the RNase MRP complex or the RNase P complex (21Schmitt M.E. Clayton D.A. Genes Dev. 1994; 8: 2617-2628Crossref PubMed Scopus (81) Google Scholar, 22Chamberlain J.R. Lee Y. Lane W.S. Engelke D.R. Genes Dev. 1998; 12: 1678-1690Crossref PubMed Scopus (206) Google Scholar). Such complex-specific protein subunits have not been described for the human enzymes yet. An alternative explanation for extensive subunit sharing is the possibility that RNase MRP and RNase P assemble into a single complex in human cells. This possibility is supported by the finding that a subset of the cellular RNase MRP and RNase P complexes has been shown to exist in such a macromolecular complex (42Lee B. Matera A.G. Ward D.C. Craft J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11471-11476Crossref PubMed Scopus (64) Google Scholar). However, in yeast, the majority of the RNase MRP and RNase P complexes can be physically separated and they function separately from each other (6Lygerou Z. Allmang C. Tollervey D. Seraphin B. Science. 1996; 272: 268-270Crossref PubMed Scopus (214) Google Scholar, 17Eder P.S. Kekuda R. Stolc V. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1101-1106Crossref PubMed Scopus (98) Google Scholar, 20Jarrous N. Eder P.S. Guerrier Takada C. Hoog C. Altman S. RNA. 1998; 4: 407-417PubMed Google Scholar, 22Chamberlain J.R. Lee Y. Lane W.S. Engelke D.R. Genes Dev. 1998; 12: 1678-1690Crossref PubMed Scopus (206) Google Scholar). The exact function and architecture of this human macromolecular complex and its relation to the individual RNase MRP and RNase P complexes remain to be elucidated and provide interesting topics for further research. We thank Drs. Cecilia Guerrier-Takada and Sidney Altman for sharing their knowledge on the purification of the RNase P holoenzyme and for their kind gift of anti-Rpp38 antibodies and Dona Wesolowski for technical assistance (Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT). The patient serum was kindly provided by Dr. Frank van den Hoogen (Department of Rheumatology, University of Nijmegen, Nijmegen, The Netherlands)." @default.
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• W2134714863 title "hPop5, a Protein Subunit of the Human RNase MRP and RNase P Endoribonucleases" @default.
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