Purification and characterization of the nuclear RNase P holoenzyme complex reveals extensive subunit overlap with RNase MRP

Abstract

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that cleaves precursor tRNA transcripts to give mature 5' ends. RNase P in eubacteria has a large, catalytic RNA subunit and a small protein subunit that are required for precursor tRNA cleavage in vivo. Although the eukaryotic holoenzymes have similar, large RNA subunits, previous work in a number of systems has suggested that the eukaryotic enzymes require a greater protein content. We have purified the Saccharomyces cerevisiae nuclear RNase P to apparent homogeneity, allowing the first comprehensive analysis of an unexpectedly complex subunit composition. Peptide sequencing by ion trap mass spectrometry identifies nine proteins that copurify with the nuclear RNase P RNA subunit, totaling 20-fold more protein than in the bacterial enzyme. All of these proteins are encoded by genes essential for RNase P activity and for cell viability. Previous genetic studies suggested that four proteins might be subunits of both RNase P and RNase MRP, the related rRNA processing enzyme. We demonstrate that all four of these proteins, Pop1p, Pop3p, Pop4p, and Rpp1p, are integral subunits of RNase P. In addition, four of the five newly identified protein subunits, Pop5p, Pop6p, Pop7p, and Pop8p, also appear to be shared between RNase P and RNase MRP. Only one polypeptide, Rpr2p, is unique to the RNase P holoenzyme by genetic depletion and immunoprecipitation studies. The large increase in the number of protein subunits over eubacterial RNase P is consistent with an increase in functional complexity in eukaryotes. The degree of structural similarity between nuclear RNase P and RNase MRP suggests that some aspects of their functions in pre-tRNA and pre-rRNA processing pathways might overlap or be coordinated.

Figure 1

RNase P activity and RPR1 RNA comigrate with a defined set of proteins in glycerol gradient velocity sedimentation. Contents of individual fractions from the final purification step, glycerol gradient sedimentation, were analyzed to locate RNase P activity and the RNA subunit, and to determine the profile of proteins that coincide with the enzyme. (A) RNase P activity was assayed by cleaving ³²P-labeled pre-tRNA^Asp to give tRNA^Asp and the 5′ leader sequence. RNA products were separated electrophoretically through 12% denaturing polyacrylamide and analyzed quantitatively by PhosphorImager. Fraction numbers are provided at the top; the input to the gradient is marked in. Sedimentation was from right (higher-numbered fractions) to left (lower-numbered fractions). (B) RNA extracted from fractions was separated on 8% denaturing polyacrylamide gels, blotted to nylon membranes, and probed with ³²P-labeled RPR1 antisense RNA to detect the RNase P RNA subunit. Amounts of RNA were estimated by quantitative comparison of the signal strength to known quantities of RPR1 sense-strand RNA prepared by in vitro transcription and blotted in parallel (not shown). (C) Protein and nucleic acid contents of fractions were visualized by denaturing PAGE in the presence of SDS and staining with silver. (Lane M) contains protein size markers with molecular mass (in kD) at right. The largest band comigrates with RPR1 RNA and does not stain with Coomassie brilliant blue in preparative gels, suggesting that it contains only the RNA subunit. The identities of proteins in lower regions of the gel were determined by excision of Coomassie-staining bands from a preparative-scale gel and exhaustive peptide analysis by ion trap mass spectrometry as described in Materials and Methods. Two bands migrating at ∼100 and 80 kD contained only Pop1p protein. The region of the two lowest visible bands contained six distinct RNase P subunits. The positions of the confirmed protein subunits relative to the polypeptide pattern are indicated at left; nomenclature is explained in the text and in Table 1.

Figure 2

All of the newly identified protein subunit candidates are encoded by essential genes. Four of the proteins that copurify with RNase P activity (Pop1p, Rpp1p, Pop4p, and Pop3p) had been identified previously as candidate RNase P subunits, and their genes had been shown to be essential. The genes for the remaining five subunits (Pop5p, Pop6p, Rpr2p, Pop7p, and Pop8p) were tested to determine whether they were essential for viability and if depletion of their gene products affected RNase P or RNase MRP function. (A) The experimental design for deleting the coding regions and substituting a gene under the regulation of a GAL1 promoter is shown schematically. Homologous recombination was used to replace one copy of each coding region in diploids with a gene for kanamycin resistance. The resulting heterodiploids were first sporulated and tested for spore viability. B shows that all tetrads segregated as two viable and two nonviable spores, the result expected for essential genes. The diagram also shows a schematic for creation of a haploid strain in which the genomic copy of the gene of interest is disrupted, and production of the gene product from a plasmid is placed under Gal control for the depletion studies shown in Fig. 3. These recombinant gene products were also all made in two versions, both of which conferred viability. One version, used for the RNA blot analyses in Fig. 3, contains only the coding region for the gene. A second version, used in the immunoprecipitation experiments in Fig. 4, also contains the 8-amino-acid Flag epitope at the carboxyl terminus.

Figure 2

All of the newly identified protein subunit candidates are encoded by essential genes. Four of the proteins that copurify with RNase P activity (Pop1p, Rpp1p, Pop4p, and Pop3p) had been identified previously as candidate RNase P subunits, and their genes had been shown to be essential. The genes for the remaining five subunits (Pop5p, Pop6p, Rpr2p, Pop7p, and Pop8p) were tested to determine whether they were essential for viability and if depletion of their gene products affected RNase P or RNase MRP function. (A) The experimental design for deleting the coding regions and substituting a gene under the regulation of a GAL1 promoter is shown schematically. Homologous recombination was used to replace one copy of each coding region in diploids with a gene for kanamycin resistance. The resulting heterodiploids were first sporulated and tested for spore viability. B shows that all tetrads segregated as two viable and two nonviable spores, the result expected for essential genes. The diagram also shows a schematic for creation of a haploid strain in which the genomic copy of the gene of interest is disrupted, and production of the gene product from a plasmid is placed under Gal control for the depletion studies shown in Fig. 3. These recombinant gene products were also all made in two versions, both of which conferred viability. One version, used for the RNA blot analyses in Fig. 3, contains only the coding region for the gene. A second version, used in the immunoprecipitation experiments in Fig. 4, also contains the 8-amino-acid Flag epitope at the carboxyl terminus.

Figure 3

Depletion of the newly identified protein subunits shows that all are essential for RNase P activity, and several are essential for RNase MRP activity. The constructs described in Fig. 2 were used to deplete Pop5p, Pop6p, Rpr2p, Pop7p, and Pop8p by shifting to growth in glucose, which impairs growth by 6 hr and reaches maximal effect on RNA processing in these strains by 12 hr. Growth of the parent haploid strain and depletion of the previously identified Rpp1p subunit were used as controls. Steady-state levels of several different RNA types were determined at 0 and 28 hr after the shift to glucose. Strain names (GAL::gene name) indicate the subunit that is under Gal control in the strain. (A) The expected products for processing of pre-tRNA^Leu3 are shown for a strain with normal or defective nuclear RNase P. Nucleolytic processing normally proceeds at the 5′ end, at the 3′ end, and at intron removal. RNase P defects lead to an increase in intron removal prior to terminal processing (+5′, +3′ intermediate). The expected products for processing of 5.8S rRNA are shown for a strain with normal participation by RNase MRP or a deficiency in RNase MRP. Mutations cause accumulation of very long (VL) and long (L) forms of 5.8S at the expense of the short (S) form. (B) Whole cell RNA samples were prepared at 0 or 28 hr after shifting to glucose to deplete the indicated subunits. RNAs were separated by electrophoresis though denaturing polyacrylamide gels, electroblotted to nylon membranes, and probed to detect the indicated RNAs. Panels are taken from a single blot exposed to the following ³²P-labeled probes (from the top): scR1 detects signal recognition particle RNA that should not be affected by either RNase P or RNase MRP defects; RNase P RNA detects both mature and precursor forms; RNase MRP RNA that detects a single form of the RNA; 5.8S rRNA detects the S, L, and VL forms; and tRNA^Leu3 detects the mature and all precursr forms. Depletion of all putative subunits strongly affects pre-tRNA processing. Effects on levels of RNase P RNA, RNase MRP RNA, and 5.8S rRNA-containing intermediates are variable with different subunit depletions and are discussed in the text.

Figure 3

Depletion of the newly identified protein subunits shows that all are essential for RNase P activity, and several are essential for RNase MRP activity. The constructs described in Fig. 2 were used to deplete Pop5p, Pop6p, Rpr2p, Pop7p, and Pop8p by shifting to growth in glucose, which impairs growth by 6 hr and reaches maximal effect on RNA processing in these strains by 12 hr. Growth of the parent haploid strain and depletion of the previously identified Rpp1p subunit were used as controls. Steady-state levels of several different RNA types were determined at 0 and 28 hr after the shift to glucose. Strain names (GAL::gene name) indicate the subunit that is under Gal control in the strain. (A) The expected products for processing of pre-tRNA^Leu3 are shown for a strain with normal or defective nuclear RNase P. Nucleolytic processing normally proceeds at the 5′ end, at the 3′ end, and at intron removal. RNase P defects lead to an increase in intron removal prior to terminal processing (+5′, +3′ intermediate). The expected products for processing of 5.8S rRNA are shown for a strain with normal participation by RNase MRP or a deficiency in RNase MRP. Mutations cause accumulation of very long (VL) and long (L) forms of 5.8S at the expense of the short (S) form. (B) Whole cell RNA samples were prepared at 0 or 28 hr after shifting to glucose to deplete the indicated subunits. RNAs were separated by electrophoresis though denaturing polyacrylamide gels, electroblotted to nylon membranes, and probed to detect the indicated RNAs. Panels are taken from a single blot exposed to the following ³²P-labeled probes (from the top): scR1 detects signal recognition particle RNA that should not be affected by either RNase P or RNase MRP defects; RNase P RNA detects both mature and precursor forms; RNase MRP RNA that detects a single form of the RNA; 5.8S rRNA detects the S, L, and VL forms; and tRNA^Leu3 detects the mature and all precursr forms. Depletion of all putative subunits strongly affects pre-tRNA processing. Effects on levels of RNase P RNA, RNase MRP RNA, and 5.8S rRNA-containing intermediates are variable with different subunit depletions and are discussed in the text.

Figure 4

Immunoprecipitations of protein subunits selectively coprecipitate RNase P and RNase MRP RNA subunits. As shown schematically in Fig. 2, Rpp1p, Pop5p, Pop6p, Rpr2p, Pop7p, and Pop8p were each expressed from plasmid gene copies in a strain where the chromosomal copy of that gene had been deleted. Recombinant proteins contained Flag epitope at the carboxyl termini. Soluble extracts were made from actively growing cultures of each strain and the parental wild type (WT) strain. Immunoprecipitation with highly specific monoclonal antibodies against the Flag epitope was followed by Northern blot analysis for the abundance of precursor and mature RNase P RNA (pre-P RNA and P RNA) and RNase MRP RNA (MRP RNA). Signal recognition particle RNA (scR1) was used as a control for an RNA that was not expected to precipitate with any of the Flag-tagged proteins. (Left) The RNA content of the input fractions; (right) RNAs found in the precipitates. RNase P and RNase MRP RNAs were not precipitated in the wild-type (WT) strain or with the Flag tag on Pop6p. All three RNAs (pre-P, P, and MRP) were precipitated with the Flag tag on Pop5p or Pop8p. Rpr2p and Pop7p gave differential coprecipitation of the RNAs. Rpr2p coprecipitated only RNase P RNAs (pre-P and P). Pop7p coprecipitated with MRP and pre-P preferentially, bringing down relatively low levels of mature RNase P RNA.

Figure 5

Sequences of the protein subunits have little similarity to bacterial or mitochondrial RNase P proteins. The amino acid sequences of the five newly identified subunits are shown, as derived from the SGD (http://genome-www.stanford.edu/Saccharomyces/). Of the five ORFs, only POP8 contains a predicted intron, composed of 75 bp between the first and second base pair of the sixteenth codon of the ORF. PCR analyses using primers flanking the intron with genomic DNA and cDNA library confirm that the POP8 cDNA gives a smaller PCR product by the expected amount. As discussed in the text, these sequences do not display strong similarities to other gene sequences in current databases and do not display notable similarities when aligned pairwise with each other or with other identified RNase P or RNase MRP proteins. Sequence features that are discussed in the text are indicated by underlining (lysines and arginines) or boldface type (lysine residues that form a short pattern in all RNase P subunits except Rpp1p).