Protein–RNA interactions in the subunits of human nuclear RNase P

Abstract

A yeast three-hybrid system was employed to analyze interactions in vivo between H1 RNA, the RNA subunit of human nuclear RNase P, and eight of the protein subunits of the enzyme. The genetic analysis indicates that subunits Rpp21, Rpp29, Rpp30, and Rpp38 interact directly with H1 RNA. The results of direct UV crosslinking studies of the purified RNase P holoenzyme confirm the results of the three-hybrid assay.

Interactions among the protein and RNA subunits ofSaccharomyces cerevisiaenuclear RNase P

“…At this time, the RNA determinants for Pop4p binding remain elusive, but they do not absolutely require the P3 subdomain of RNase P (unpublished data) and it might be that Pop4p is interacting with multiple sites in the RNA. A recent three-hybrid test of human RNase P RNA (H1 RNA) identified possible interactions with the human homologs of Pop4p (Rpp29p), Rpp1p (Rpp30p), and Rpr2p (Rpp21p) (56). The human results are in stark contrast to our results in yeast; the human three-hybrid study did not identify an interaction between H1 RNA and human Pop1p whereas we could not detect interactions between RPR1 RNA and either Rpp1p or Rpr2p.…”

Purification and characterization of Rpp25, an RNA-binding protein subunit of human ribonuclease P

“…Immunoprecipitation of HeLa RNase P+ HeLa RNase P purified through the glycerol gradient step was subjected to immunoprecipitation analysis using Protein A beads coupled with sera obtained from rabbits immunized with recombinant Rpp25 and Rpp21, as well as preimmune sera, as described in Materials and Methods+ Supernatant fraction (top panel) and immunoprecipitated fraction (bottom panel) were assayed using 32P labeled pSupS1 (Krupp et al+, 1985) as substrates+ Aliquots were taken after 2, 4, 6, 8, and 10 min incubation, and the products of the reaction were analyzed on an 8% denaturing polyacrylamide gel+ Lane 1: pSupS1 incubated alone, for 10 min; lanes 2-6: samples treated with anti-Rpp25 antibodies; lanes 7-11: samples treated with anti-Rpp21 antibodies; lanes 12-16: samples treated with preimmune sera+ The position of the reaction products are indicated: S ϭ pSupS1; 59 ϭ 59 leader sequence; 39 ϭ tRNA moiety+ FIGURE 5. UV-crosslinking and gel shift assays+ A: 9% polyacrylamide/SDS gel+ 32 P-labeled Sty RNA was incubated alone (lane 5), with recombinant Rpp25 (lane 1), Rpp20 (lane 2), Rpp40 (lane 3), or RNase P fraction purified through the glycerol gradient step (lane 4), as described in Materials and Methods+ After UV irradiation for 4 min at room temperature, the samples were loaded onto a 9% polyacrylamide/SDS gel+ The gel was dried and exposed to autoradiography+ Specific complexes can be seen in lanes 1 and 4+ B: 5% nondenaturing polyacrylamide gel+ 32 P-labeled H1 RNA (0+04 pmol) was incubated for 10 min in the presence of increasing concentration of Rpp25 (0, 0+3, 0+6, 1+2, and 2+5 pmol), without (lanes 1-5), or with (lanes 6-9) the His tag+ 32 P-labeled pTyr (0+5 pmol) was incubated for 5 min in the presence of increasing concentration of Rpp25 (0+6, 1+2, 2+5, and 5 pmol), without (lanes 10-13) or with (lanes 14-17) the His tag+ After incubation, the samples were loaded on a 5% polyacrylamide gel as described in Materials and Methods+ Multiple complex bands can be seen, as indicated by the arrows+ there are no RGG boxes in human Rpp25, the nonspecific binding exhibited by Rpp25 is reminiscent of the characteristics of RBPs like the RGG box-containing proteins (Burd & Dreyfuss, 1994)+ Moreover, such a functional role might prove redundant in some instances and could help rationalize the absence of Rpp25 homologs in some eukaryotes+ Some of the protein subunits of human RNase P were shown to interact with the cognate RNA subunit by using a three-hybrid assay and UV crosslinking (Jiang et al+, 2001)+ As part of a complementary approach, experiments are now in progress to purify the various protein subunits of the human RNase P complex in soluble form and investigate if any of them can bind H1 RNA in vitro, either alone (like Rpp25) or in some combination with other factors+…”

“…Human RNase P was purified through the glycerol gradient fractionation and Mono Q FPLC steps of the procedure described elsewhere (Eder et al+, 1997)+ Fractions constituting the peak of enzyme activity after glycerol gradient and MonoQ chromatography were FIGURE 1. cDNA sequence and translated polypeptide sequence for Rpp25+ The nucleotide sequence is numbered from the start of the total sequence indicated+ The amino acid sequence is numbered from the first methionine residue and is shown in boldface letters+ The portions of the peptide sequences that correspond to the tryptic peptide fragment derived from authentic Rpp25 are underlined+ Rpp25, a protein subunit of human RNase Psubjected to western blot analysis using anti-Rpp25 and Rpp38 antibodies+ The finding that strong crossreactivity to anti-Rpp25 antisera was observed only in the peak fractions confirmed that Rpp25 was associated with human RNase P activity (Fig+ 3)+ Western blot analysis were also carried out using antibodies to Rpp20, Rpp21, Rpp29, Rpp30, Rpp40, and hPop1: results confirmed the copurification of these proteins with RNase P activity (data not shown)+ Polyclonal antibodies raised against recombinant Rpp25 were also tested for their ability to precipitate catalytically active RNase P complexes from partially purified preparations of RNase P+ Immunoprecipitation of active holoenzyme was observed using anti-Rpp25 antibodies+ Similar results were obtained using antiRpp21 antibodies, whereas preimmune sera failed to immunodeplete RNase P activity (Fig+ 4)+ These results led us to investigate whether the purified, his-tagged Rpp25 could bind to H1 RNA, the RNA subunit of human RNase P+ Interactions of Rpp25 with the RNA subunit of human RNase P and ptRNA substrates Purified recombinant Rpp25 protein, as well as Rpp20 and Rpp40, were incubated with 32 P-labeled H1 RNA, and a fragment of H1 RNA, the first 74 nts, containing the P3 domain (Chen & Pace, 1997;Jiang et al+, 2001; referred to here as Sty RNA) and the mixture was subjected to short-wave UV irradiation+ The products of the reaction were analyzed on a 9% SDS/ polyacrylamide gel (Fig+ 5A)+ Specific complexes with Sty RNA can be seen in Figure 5A, lane 1; higher molecular weight complexes can be seen in lane 4, with glycerol gradient enzyme, indicating that other protein subunits also interact with the holoenzyme+ No interaction was observed with Rpp20 (Fig+ 5A, lane 2), nor Rpp40 (Fig+ 5A, lane 3)+ Using H1 RNA as the RNA species, with purified Rpp25 the only complex observable migrates near the stacking/ separation gel boundary (data not shown)+ A similar result is seen with an RNase P fraction purified through the glycerol gradient step (data not shown)+ Accordingly, Rpp25 interactions with several RNAs were then assayed by performing gel shift assays+ Rpp25 and RNA samples were incubated for 10 min and then loaded directly on a 5% nondenaturing gel (see Materials and Methods)+ The RNAs tested included H1 RNA and an RNase P substrate, the precursor to E. coli tRNA Tyr (pTyr; Guerrier-Takada et al+, 1989)+ These experiments were also carried out with Rpp25 in which the His-tag was removed+ The interaction of Rpp25 with the various RNAs tested is not influenced by the absence or presence of a His-tag in Rpp25 (Fig+ 5B)+ As can be seen, multiple complexes are detected; these complexes are still present even in the presence of 5 mM DTT in the reaction mix (data not shown)+ We have also observed complexes of Rpp25 with M1 RNA (the RNA subunit of E. coli RNase P), and with an non-RNase P-related RNA fragment (data not shown)+ It has recently been reported (Jarrous et al+, 2001) that Rpp14 and Rpp21, two protein subunits o...…”

“…Proteins in fractions obtained after glycerol gradient and MonoQ FPLC fractionation procedures were separated on 12% polyacrylamide/SDS gel, electrotransferred to a nitrocellulose membrane, and immunoblotted with a 1:100 dilution of sera as described (Jiang et al+, 2001)+ As a secondary antibody, a 1:5,000 dilution of anti-rabbit mouse IgG antibody (Vector Laboratories) was made in the same buffer and incubated for 60 min+ Blots were washed with TNT and TN (Jiang et al+, 2001) and antibody-antigen complexes were visualized using the ECL-Plus detection system (AmershamPharmacia), following the manufacturer's instructions+…”

“…HeLa nuclear RNase P purified through the glycerol gradient step or purified recombinant Rpp25 were incubated at 37 8C in the presence of labeled H1 RNA, an H1 RNA fragment containing the P3 domain, or a precursor tRNA substrate in 20 mM HEPES-KOH, pH 8+0, 1 mM MgCl 2 , 200 mM NaCl, 5% glycerol (HGMN buffer)+ After 5 min, the samples were either placed on a sheet of saran wrap over a short-wave UV transilluminator and irradiated for 4 min at room temperature (Jiang et al+, 2001 ), or loaded onto a 5% polyacrylamide gel (20 ϫ 20 ϫ 0+1 cm) with 20 mM HEPES-KOH, pH 8+0, 1 mM MgCl 2 as the running buffer+ Electrophoresis at 30 mA was carried out at 4 8C for 2 h, with continuous recirculation of the buffer+ Results were quantitated using a Fuji Phosphorimager+…”