Biochirnica et Biophysica Acta, 1087 (1990) 31-38
31
Elsevier BBAEXP 92156
Class II ribonuclease H comigrates with, but is distinct from, the third largest subunit of calf thymus RNA polymerase I Heiner Vonwirth
1, P e t e r
Frank
1, C l a u d e
Kedinger 2 and Werner Biisen 1
1 Lehrstuhlfiir Allgemeine Genetik, Biologisches Institut, Tiibingen (F.R.G.) and e Laboratoire de Genetique Moleculaire des Eucaryotes du CNRS, Strasbourg (France)
(Received 6 February1990)
Key words: RibonucleaseH; RNA polymeraseI; Autoimmuneantibody It has been reported (Iborra et al. (1979) J. Biol. Chem. 254, 10920-10924) that the third and the fifth largest subunit of yeast RNA polymerase I exhibit ribonuclease H activity. The authors suggested that the third largest subunit is identical with the chromatin-associated ribonuclease H49 , the putative yeast equivalent of bovine ribonuclease H lib. Although the third largest subunit of calf thymus RNA polymerase I and ribonuclease H Ilb display nearly identical molecular masses under denaturing conditions, serological analysis reveals that, in contrast to their counterparts in yeast, these mammalian proteins are distinct entities. Interestingly, sera from some patients with mixed connective tissue disease which contain antibodies directed against RNA polymerase I, also react with ribonuclease H Ilb epitopes. This observation suggests that a protein displaying ribonuclease H lib antigenicity could be associated with RNA polymerase I. Additional indications supporting this conclusion are discussed.
Introduction Enzymes with the specificity of a ribonuclease H (EC 3.1.13.2) first discovered by Hausen and Stein [1], are widespread among pro- and eukaryotes (for a review see Ref. 2). In calf thymus, three distinct ribonuclease H activities, named H I, H IIb and H IIa, are found. Whereas H I and H IIb display ribonuclease H specificity, H IIa lacks this specificity [3-7]. Enzymes I and IIb were purified and polyclonal antibodies were raised against these protein fractions. Antibodies directed against ribonuclease H I inhibit only this enzyme activity [8,9]. Antibodies raised against ribonuclease H IIb deplete enzyme extracts from the antigenic protein band and enzyme activity [6]. Both enzymes cannot only be differentiated at the serological level but also by their physical and biochemical parameters, including their mode of action [3-10]. Ribonuclease H I, an endonuclease, and H IIb, presumably an exonuclease, seem
Abbreviations: SDS, sodium dodecyl sulfate; PMSF, Phenylmethylsulfonyl fluoride; DTr, Dithiothreitol; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethane sulfonicacid; MCTD, mixed connectivetissue disease. Correspondence: W. Btisen, Lehrstuhl fiir AllgemeineGenetik, Bioiogisches Institut, Universit~it Ttibingen, Auf der Morgenstelle 28, D-7400 Tiibingen,F.R.G.
to play different functional roles in vivo, as suggested by observations that their activity levels are elevated at different times after the stimulation of resting bovine lymph node cells with concanavalin A. Ribonuclease H IIb activity parallels the increase in RNA synthesis, whereas ribonuclease H I increases concomitant with the rise in DNA synthesis [11]. Further indications for a relationship between the class II ribonuclease H and transcription have been reported in the literature. Tsukada et al. [12] and Sawai et al. [13] found that the liver nuclei of thioacetamide treated rats show a parallel increase of RNA polymerase I (EC 2.7.7.6) and class II ribonuclease H activity. The class I ribonuclease H and RNA polymerase II activity remained nearly unaffected by this treatment. This does not exclude a possible link between the other RNA polymerase species (II and III) and class II ribonuclease H, but it stresses the interaction between R N A polymerase I and class II ribonuclease H. Further support for this link was derived from the findings of Iborra et al. [14-16] who reported that the third and the fifth largest subunit of RNA polymerase A (I) of yeast possesses ribonuclease H activity. This association has not been confirmed in yeast so far, nor has it been shown to exist in higher eukaryotes. Because of the importance of this finding and the possible functional implications, we have asked whether RNA polymerase I, and in particular the third largest
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32 subunit of calf thymus RNA polymerase I displays ribonuclease H lib antigenicity. Here we report, that the molecular masses of the ribonucleases H lib were comparable to those reported for the third largest subunits of the corresponding RNA polymerases I in calf thymus and mouse cells. In more or less purified calf thymus samples we found a protein with ribonuclease H lib antigenicity coeluting with RNA polymerase I. However, serological analysis revealed that the calf thymus protein displaying ribonuclease H lib antigenicity and the third largest subunit of calf thymus RNA polymerase I were distinct from each other. We discuss indications for a possible specific binding of class II ribonuclease H to RNA polymerase I in mammalian systems. Materials and Methods
Assay for ribonuclease H. The standard [3H]RNA. DNA hybrid was prepared as described in previous communications [3]. The specific activity of the hybrid was 66 d p m / p m o l ribonucleotide. Ribonuclease H lib assays were carried out as described elsewhere [3,6]. Assay for RNA polymerase. RNA polymerase activity was measured according to Stein and Hausen, and Dahmus [17,18] with minor modifications. A final assay volume of 100 #1 containing 30 mM Tris-HC1 (pH 7.8), 50 mM (NH4)2SO4, 2 mM MnC1 z, 1 mM dithiotreitol (DTT), 1 mM each of ATP, CTP and GTP, 0.1 mM [3H]UTP (400 d p m / p m o l ) , 12.5 /xg of non-denatured calf thymus DNA, and various amounts of enzyme solution was incubated for 10 rain at 37 ° C. The reaction was stopped by addition of 100 /~1 8% trichloroacetic acid and incubation for 10 rain on ice. The resulting precipitate was collected on a Na4P20 7saturated nitrocellulose filter (Millipore), washed, dried, and counted in a liquid scintillation counter. 200 cpm correspond to 1 pmol ribonucleotides incorporated. Protein determination. Protein concentration was determined according to Bradford using bovine serum albumin as a standard [19]. SDS-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in the presence of SDS was carried out in 1-nun-thick slab gels according to Laemmli [20]. After electrophoresis, the gels were processed for silver staining [21,22]. Molecular masses were determined as described [6]; myosin (205 kDa) was used as an additional marker. Immunological detection of proteins. Western blotting experiments were carried out as described [6]. The nitrocellulose sheets were blocked with bovine serum albumin and incubated overnight with rabbit serum (either control- or antiserum) or human-autoimmune serum as indicated in the individual experiments. Binding of IgG was visualized by incubation with peroxidase-conjugated goat-anti-rabbit or goat-anti-human IgG (H +
L) followed by peroxidase reaction.
Preparation of monospecific antibodies. Antibodies bound to distinct protein bands were eluted and used directly for immunological analysis as described previously [6]. Preparation of nuclear extracts. Nuclear extracts from mouse Ehrlich Ascites and HeLa cells were prepared according to D i g - a m et al. [23]. Cells were grown to a density of about 6 . 1 0 5 cells per ml. The cells were harvested from the culture medium by centrifugation for 10 min at 200 x g and 4°C. The cell pellet was washed twice with cold N a C 1 / P i (phosphate-buffered saline according to Dulbecco [24], but without Ca 2+ and MgZ+), and suspended in five packed-cell volumes of buffer A (10 mM Hepes (pH 7.9), 1.5 mM MgC12, 10 mM KC1 and 0.5 mM DTT) and allowed to stand for 10 min at 4 ° C . The suspension was centrifuged for 10 min at 200 X g and 4 ° C and the pellet was resuspended in two packed-cell volumes of Buffer A. The cells were lysed by 15 strokes with a Dounce homogenizer and centrifuged for 10 min at 200 x g and 4 o C. The supernatant was decanted and processed for preparation of the S100 fraction exactly as described [23]. The pellet was again centrifuged (20 min at 25 000 x g and 4 ° C) and the sediment of the second centrifugation step suspended in buffer C (20 mM Hepes (pH 7.9), 25% glycerol, 0.42 M NaC1, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT) (3 ml/109 cells) using a Dounce homogenizer. The suspension was stirred gently for 30 min at 4 ° C and centrifuged for 30 min at 2 5 0 0 0 x g and 4 ° C . The supernatant was dialysed against 50 volumes of buffer D (20 mM Hepes (pH 7.9), 20% glycerol, 0.1 M KC1, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT). The dialysate was centrifuged as above and the clear supernatant frozen as aliquots in liquid nitrogen and stored at - 7 0 ° C. The protein concentrations of the S100 fractions were between 2 and 8 m g / m l and those of the nuclear extracts between 5 and 10.4 mg/ml. Anti-calf thymus ribonuclease H lib-affinity chromatography. The anti-calf thymus Ribonuclease H IIb affinity matrix was prepared as described [6]. The enzyme solution was incubated overnight at 4 ° C with the affinity matrix. The suspension was filtered through a Btichner funnel and the flow through collected. Ribonuclease H activity was determined in the enzyme solution before adsorption to the matrix and in the flowthrough fraction.
Preparation of calf thymus crude extracts and separation of RNA polymerases. Calf Thymus crude extracts were prepared as described previously [6]. The crude extract from 75 g frozen calf thymus was diluted 10-fold with buffer B (30 mM Tris/HC1 (pH 7.8), 2 mM EDTA, 0.1% 2-mercaptoethanol (v/v), 30% glycerol (w/v)) and adsorbed onto DEAE-cellulose (140 g of preswollen DE52, Whatman). The matrix was washed
33 extensively with buffer B, loaded onto a column (4.9 cm2 × 40 cm), and the proteins were eluted with a linear salt gradient (0-1000 mM KC1 in buffer B). Fractions of 2 ml were collected and the RNA polymerase activity of 20 gl of the individual fractions was determined, without and with the addition of a-amanitin (2 # g / m l assay). Under these conditions RNA polymerase II activity is strictly inhibited while RNA polymerase I activity is not affected. Calf thymus RNA polymerase L" enzyme and specific antiserum preparation. Calf thymus RNA polymerase I (originally named AI) was purified to apparent homogeneity, as described previously [25]. The experiments reported hereafter were performed with the glycerolgradient fraction (GG) concentrated to 10 mg protein per ml by vacuum-concentration through a Sartorius dialysis bag. Antibodies were raised against an independent preparation of RNA polymerase I fraction GG [26] which gave a SDS-polyacrylamide gel electrophoresis pattern indistinguishable from that of the fraction used in the immunoblot analysis. Calf thymus ribonuclease H IIb: enzyme and specific antiserum preparation. Calf thymus ribonuclease H IIb was purified to apparent homogeneity as described previously [6]. The enzyme fraction used in this report corresponds to the most purified enzyme fraction (fraction 7) [6]. Antibodies were those used in the previous study [6].
A utoimmune sera. Sera used were from clinically identified mixed connective tissue disease (MCTD) patients. They were also shown to contain antibodies directed against U1-RNP (R. Liihrmann, Marburg, personal communication). Chemicals and isotopes. All chemicals used were reagent grade and obtained from Boehringer Mannheim, Merck, Serva and Sigma. [3H]UTP was from Amersham International (U.K.). Ribo- and deoxyribonucleoside triphosphates, native calf thymus DNA, protein markers, E. coli RNA polymerase, yeast RNA and PMSF were obtained from Boehringer (Mannheim, F.R.G.). Results
Correspondence of the molecular masses of ribonuclease H lib and the third largest subunit of RNA polymerase I in mammalia Although the biological function of a eukaryotic class II ribonuclease H is not known, several lines of evidence point to a function during the transcription process, as indicated in the introduction. Unfortunately these findings, particularly the association of ribonuclease H and RNA polymerase I, have been neither confirmed nor disproven in yeast or in any other eukaryotic system. So far we have failed to find ribonuclease H enzymatic activity in association with mammalian RNA
HeLa A
A
C
Calf Thymus C
116-94-67--
6!6-o52-45. 40--
25--
1 2
3
1
2
3
~
2'
3'
1'
2'
3'
Fig. 1. Mammalian ribonuclease H Ilb-antigenicity is associated with two fractions. HeLa cell nuclear extracts and calf thymus crude extracts were adsorbed to DEAE-CeUulose and the 200 mM KCI-DEAE-ceUulose eiuates, together with the DEAE-cellulose flow-through fractions, were analysed by immunoblotting using the antiserum directed against calf thymus ribonuclease H IIb (A) and the control serum (C). Proteins were separated on 12% polyacrylamide gels and processed for immunoblotting as described in Materials and Methods. Antiserum (A) was diluted 1:1000, control serum (C) 1:500. Lanes 1, 2 and 3 correspond to 25 gg of protein of the HeLa cell nuclear extract, of the 200 mM KC1-DEAE-cellulose eluate, and of the DEAE-cellulose flow-through, respectively. Lanes 1', 2' and 3' correspond to 10 #g of protein of calf thymus crude extract, to 100 #g of protein of the 200 mM KC1-DEAE-cellulose eluate, and to 10 #g of protein of the DEAE-cellulose flow-through. The slightly different migration behavior of the antigenic band in lane 3' in comparison to that in lane 2' is due to band curvature at the margin of the gel.
34 polymerase I, but the availability of specific antisera against calf thymus ribonuclease H lib and RNA polymerase I, and of highly purified protein fractions of both enzymes allowed us to explore the situation in mammalian systems at the serological level. In a previous study we identified calf thymus ribonuclease H IIb as most likely a monomeric enzyme with apparent molecular masses of 45 and 52 kDa under native and denaturing conditions, respectively [6]. Interestingly, the 52 kDa value corresponds very well to the 51 kDa reported for the third largest subunit of calf thymus RNA polymerase I [26]. To investigate whether this correlation also holds true for other mammalian systems, proteins of HeLa cell and mouse nuclear extracts were processed for western blotting. Fig. 1 (lane 1, A) shows that the anti-calf thymus ribonuclease H IIb-serum cross-reacted specifically and predominantly with a 61 kDa protein band but also with a minor 52 kDa protein band of the HeLa cell nuclear extract. This finding has been confirmed by immunoblots using a monospecific antibody isolated from affinity-purified calf thymus ribonuclease H lib (see Materials and Methods) (not shown, Ref. 32). Depletion experiments with fractions containing exclusively ribonuclease H lib activity (see Materials and Methods) revealed that the antigenic protein bands are quantitatively and the enzyme activity is almost quantitatively retained by the anti-calf thymus ribonuclease H lib-affinity matrix (not shown, Ref. 32). From these data we conclude that ribonuclease H IIb activity must be associated with the 61 and 52 kDa-proteins. Gel filtration experiments revealed a native molecular mass of 45 kDa (not shown, Ref. 32), the same value which was determined for the calf thymus enzyme [6]. We therefore suggest that this HeLa enzyme activity is the human homologue to calf thymus ribonuclease H lib [32]. A ribonuclease H with properties
l 3.Mr
similar to our calf thymus ribonuclease H IIb was recently isolated from HeLa cells [33]. The final preparation was not homogeneous, but it is striking to see that the two most prominent protein bands of the most purified fraction show molecular masses between 50 and 60 kDa. There is no report in the literature on the molecular mass of the third largest subunit of human RNA polymerase I, but it is interesting to note that the molecular mass for the third largest subunit of mouse RNA polymerase I has been reported to be 61 kDa under denaturing conditions [27]. Experiments performed with mouse nuclear extracts also revealed a 61 kDa-protein to be crossreactive (not shown, Ref. 32). These initial, preliminary results were compatible with the idea that the third largest subunit of mammalian RNA polymerase I is ribonuclease H lib.
Less purified fractions of RNA polymerase I display ribonuclease H lib antigenicity DEAE-cellulose chromatography separates ribonuclease H lib and RNA polymerase I activities from each other (the former one stays in the flow-through fraction whereas the latter one is retained on this matrix). If ribonuclease H IIb is identical with the third largest subunit of RNA polymerase I, the antibody directed against calf thymus ribonuclease H lib should reveal antigenicity not only to proteins present in the flow-through, but also to proteins bound to the matrix. To address this question, proteins of the crude extract (Fig. 1 lanes 1, 1'), the DEAE-cellulose flow-through (lanes 3, 3'), and the fraction bound to the matrix (lanes 2, 2') were processed for immunoblotting. As can be seen in Fig. 1, all fractions of HeLa cells and calf thymus tissue displayed ribonuclease H lib antigenicity in association with proteins of the expected size. According to the protein recovery from the DEAE-cel-
0,0 0,05 0,1 0,2 0,5 1,0 0,7 0,5 0,45 0/,5 0,35
RNA Pol. I Activity
116-94-67-60-40--
25--
33
35 3"7 39
M
43 45 47 49 51
53
Fraction Number
Fig. 2. A protein with fibonucle~e H l i b anfigenicity coelutes on DEAE-ceUulose with R N A polymer~se ! of calf thymus. C~lf thymus crude
extract was adsorbed to DEAE-cellulose,the proteins were eluted with a linear salt gradient and the individual fractions were tested for RNA polymerase activity. Proteins of the indicated fractionswere separated on a 12% polyacrylamidegel and processed for immunoblottingusing the anti-ribonucleaseH lib-serum. The a-amanitininsensitiveRNA polymeraseactivityis indicated in relativeunits along the top of the figure.
35 lulose chromatography (not shown), the intensities of the antigenic protein bands suggest a more or less equal distribution of the antigens between the fraction binding to DEAE-cellulose and the flow-through fraction in HeLa cells, and only about 5% antigenicity associated with the DEAE-cellulose-retained fraction from calf thymus (under the conditions used, the DEAE-cellulose flow-through fraction was quantitatively depleted from ribonuclease H I, [3,8] (not shown)). This result is remarkable because ribonuclease H lib enzymatic activity is found only in the DEAE-cellulose flow-through fraction [3,6]. In a next step we wanted to see whether the proteins binding to DEAE-cellulose and displaying ribonuclease H lib antigenicity coeluted with RNA polymerase I activity. We adsorbed calf thymus crude extract to DEAE-cellulose, eluted the bound proteins with a linear salt gradient, and tested the gradient fractions for RNA polymerase activity as described in Materials and Methods. We identified the fractions containing RNA polymerase I and II activity and tested these fractions for crossreaction with the polyclonal antibody against ribonuclease H lib by immunoblotting. Fig. 2 shows an immunoblot throughout the (a-amanidn insensitive) RNA polymerase I activity peak. As can be seen, antigenicity coelutes with the beginning and the left half of the RNA polymerase I activity peak. Under the assumption that no inhibitors interfere with RNA polymerase activity and that the measurable RNA polymerase activity reflects the distribution of RNA polymerase molecules, one could state that ribonuclease H IIb antigenicity is restricted to a subfraction of RNA polymerase I. Indications for more than one form of RNA polymerase I are reported in the literature [27,28]. Antibodies directed against calf thymus RNA polymerase I recognize ribonuclease H lib To further prove the postulated association of ribonuclease H IIb and RNA polymerase I, we performed immunoblots with fractions of several different purification steps of calf thymus ribonuclease H lib and polyclonal antibodies directed against this enzyme ( a l l IIb) and calf thymus RNA polymerase I (aPol I). Fig. 3 shows an example of such an experiment performed with the most purified ribonuclease H lib (fraction 7, Ref. 6). A strong antigenic response to the 52 kDa ribonuclease H lib protein was detected with antiserum directed against ribonuclease H lib, as well as with antiserum raised against RNA polymerase I. The specificity of this reaction is underlined by the fact that only about 5% of the polypeptides comprised by the most purified ribonuclease H lib fraction have a molecular mass of 52 kDa under denaturing conditions (Fig. 3, lane t). The remaining peptides of lower molecular masses correspond to degradation products of the native enzyme [6]. Thus, both sera react specifically with
lO~.Mr ~HIIb . P o l I C
116
t ..........
9Z,~ 67~
60
524~-:_ 40--
-
25--
Fig. 3. Immunoblot analysis of purified calf thymus ribonuclease H lib. Purified calf thymus ribonuclease H IIb (4 #g of protein of fraction 7, Ref. 6) was separated and processed for immunoblotting. aH lib (anti-ribonuclease H lib serum, 1:1000), ttPol I (anti-RNA polymerase I-serum, 1:1000), C (control serum, 1:500), t (silver staining of 4/~g of purified ribonuclease H IIb).
the intact 52 kDa ribonuclease H IIb polypeptide chain. We conclude that even the highly purified calf thymus RNA polymerase I fraction used to raise antibodies must have contained ribonuclease H lib antigenic proteins. Human autoimmune sera react specifically with ribonuclease H lib It is known from the literature that sera from patients with the mixed connective tissue disease (MCTD) detect RNA polymerase I specifically, and not the RNA polymerases II and III. Immunoblotting analysis has shown that some of these sera contain antibodies directed against the third largest subunit of RNA polymerase I [29]. Thus, if there is an identity of this subunit with the ribonuclease H lib protein, highly purified ribonuclease H lib should be also recognized by some sera from MCTD patients. We examined 14 different sera from MCTD-patients and looked for crossreaction of these sera with highly purified calf thymus ribonuclease H IIb. In 7 out of 14 sera we found a clear crossreaction with the ribonuclease H lib-specific 52 kDa-protein band. Fig. 4 shows immunoblots performed with two of the cross-reacting autoimmune sera. Strong crossreactivity was found in association with either the 52 kDa protein band alone (left) or the 52 and the 45 kDa protein bands (right). These data suggest that ribonuclease H lib serves as an antigen in at least some MCTD-patients. The third largest subunit of RNA polymerase I is distinct from ribonuclease H IIb The data presented above do not discriminate between the possibilities that the third largest subunit of
36 RNA polymerase I and ribonuclease H IIb are associated with one and the same polypeptide chain, that ribonuclease H IIb is specifically associated with RNA polymerase I but distinct from the third largest subunit, or that ribonuclease H lib simply copurifies with RNA polymerase I. Highly purified samples of calf thymus RNA polymerase I made a decision possible. Fig. 5 presents immunoblots performed with highly purified calf thymus RNA polymerase I (fraction GG, see Fig. 5 of Ref. 25 and Fig. 2 of Ref. 26; lane 1) and highly purified calf thymus ribonuclease H lib (fraction 7, Ref. 6; lane 2). As can be seen in lane 1, aPol I, the RNA pol I antiserum detected the four largest RNA polymerase I subunits with molecular masses of 205, 127, 53, and 43 kDa under our conditions. On the contrary, the ribonuclease H lib antiserum did not recognize any of the RNA pol I subunits (lane 1, aH IIb). This finding proves that the third largest subunit of RNA polymerase I and ribonuclease H lib are distinct from each other. At the first glance this seems to be a contradiction to the result presented in Fig. 3, in which it was shown that the RNA pol I antiserum cross-reacted with the most purified ribonuclease H IIb fraction. To resolve this question we isolated, from the RNA pol I antiserum, monospecific antibodies directed against the ribonuclease H IIb specific 52 kDa protein band (aPol Im52). AS can be seen in Fig. 5 lanes 1 and 2, aPol Im52, this subfraction of the RNA pol I serum does recognize the ribonuclease H IIb specific 52 kDa protein band but neither the third largest subunit of RNA polymerase I nor any other subunit. For that reason we state that the
MCTD-Sera 116-94-67--
526043-= 40--
25--
Fig. 4. Immunoblot analysis of purified calf thymus ribonuclease H lib by use of autoimmune sera. Purified calf thymus ribonuclease H IIb (4 /~g of protein, see Fig. 3) was separated and processed for immunoblotting. MCTD-sera were diluted 200-fold.
103Mr
,PolI ,tH2~b C
205--
.~HIIb
,,Po/ Im52
m
116--
67-GO--
45--
I
I
1
I
2
I
2
Fig. 5. Immunoblot analysis of purified calf thymus RNA polymerase I and calf thymus ribonuclease H lib with distinct antisera. Purified calf thymus RNA polymerase I (12 #g of protein) (lanes 1) and purified calf thymus ribonuclease H lib (4 #g of protein, see Fig, 3) (lanes 2) were separated on 8% SDS-polyacrylamide gels and processed for immunoblottings, aPol I (anti-RNA polymerase l-serum, 1 : 1000), aH IIb (anti-ribonuclease H IIb-serum, 1:1000), C (control serum, 1 : 500), aPol Ira52 (monospecific antibodies isolated out of the polyclonal anti-RNA polymerase I-serum after binding to the affinity purified ribonuclease H lib specific protein band with a molecular mass of 52 kDa).
purified RNA polymerase I fraction used to raise antibodies must have contained a protein with ribonuclease H IIb antigenicity. The highly purified RNA polymerase I fraction used in the experiments shown in Fig. 5 must have lost this protein during the purification procedure. These results indicate that, depending on the preparation, highly purified RNA polymerase I can contain an additional protein with ribonuclease H IIb antigenicity, and that the third largest subunit of RNA polymerase I is distinct from the protein with ribonuclease H lib antigenicity. Discussion
More than 10 years ago Huet et al. [14,15] showed that highly purified yeast RNA polymerase I displays ribonuclease H activity. Moreover, Iborra et al. [16] were able to show that the third largest subunit of RNA polymerase I is identical with the chromatin associated ribonuclease H49. These results, together with the data presented by Biisen et al., Tsukada et al., and Sawai et al. (see Introduction), which showed that class II ribonuclease H activity is regulated with transcription and RNA polymerase I activity, respectively (Refs. 11-13), are in favor of the interpretation that the association
37 found by Huet et al. [14,15] is biologically relevant. It is therefore more than surprising that these interesting results were neither confirmed nor disproven in yeast or in any other eukaryotic system. In this paper we could show, by using different types of antibodies specific for either RNA polymerase I or ribonuclease H IIb, that in calf thymus the third largest subunit of RNA polymerase I and ribonuclease H lib correspond to two distinct polypeptide chains. This is in variance to what has been reported for yeast RNA polymerase I. Nevertheless, proteins showing ribonuclease H IIb antigenicity can be found in highly purified fractions of RNA polymerase I. This could mean that RNA polymerase I and ribonuclease H lib bind specifically to each other and that both proteins may copurify through several purification steps. This conclusion is supported by the following observations: (1) Whereas ribonuclease H lib does not bind to DEAE-cellulose, a protein with ribonuclease H lib antigenicity coelutes with RNA polymerase I from this matrix. (2) The findings that all sera of MCTD patients recognize RNA polymerase I specifically [29] and that some MCTD-sera - as shown in this report - recognize ribonuclease H lib in addition, are compatible with the notion that some patients develop sera against a RNA polymerase I-ribonuclease H IIb complex. In this context it should be mentioned that anti-Sm autoantibodies, prototype antibodies reacting with antigens of RNA-protein complexes and often produced by MCTD-patients, are reported to detect specifically yeast ribonuclease H55 [30], the enzyme of a proteinase-deficient yeast strain, believed to correspond to yeast ribonuclease H49, characterized by Iborra et al. [16,31]. (3) Recently, we separated at least two forms of RNA polymerase I in the protozoan Crithidia fasciculata, from which only one displays ribonuclease H IIb antigenicity. (4) Coelution of this antigen with RNA polymerase I can be followed through the whole purification procedure, including gel filtration under 0.1 M (NH4)2SO 4 (Ref. 32; K~Sck, J. et al., unpublished data; Vonwirth, H. et al., unpublished data). Roberge and Bradbury studied the interactions between nascent R N A and RNA polymerase I-transcribing complexes in HeLa cell nucleoli by photoaffinity labeling [34]. They observed a specific labeling of the two largest subunits of RNA polymerase I and of a 52 kDa protein band. In the light of the data presented by Huet et al. [14,15] and Iborra et al. [16] they speculate that the photoaffinity-labeled 52 kDa protein band is the human equivalent of the yeast third largest subunit of RNA polymerase I and therefore also of the yeast ribonuclease H. As an alternative, they propose that the 52 kDa protein band is not a subunit of RNA polymerase I but a distinct polypeptide intimately bound to the transcribing polymerase. Our data do not support
their first suggestion. However, our results do not allow to differentiate whether the 52 kDa protein band is the third largest subunit of HeLa cell RNA polymerase I or the human equivalent of our protein displaying ribonuclease-H lib antigenicity or another unknown protein. All data presented in this paper and others (see Introduction) render unlikely a fortuitous association of ribonuclease H activity a n d / o r ribonuclease H IIb antigenicity with RNA polymerase I. Nevertheless, final proof has to await for functional correlations during the RNA polymerase I transcription process. Since ribonuclease H activity could not be demonstrated for the protein displaying ribonuclease H lib antigenicity, we have to be also aware of the possibility that an enzymatic activity different from ribonuclease H activity is important for its function in vivo. Answers can be expected from studies using highly purified ribonuclease H lib, RNA polymerase I with and without the associated protein with ribonuclease H IIb-antigenicity and the specific antibody against ribonuclease H IIb in in vitro systems for proper initiation or termination of RNA polymerase I transcription [35,36].
Acknowledgements The advice and support of Prof. Dr. W. Seyffert is gratefully acknowledged. We are indebted to Prof. Dr. R. Liihrmann (Marburg) for kindly supplying us with the diverse sera of MCTD patients, for critically reading the manuscript and for helpful advice. Thanks also to Prof. I. Grummt (Wtirzburg) for the kind gifts of nuclear extracts of mouse Ehrlich ascites cells and purified fractions of mouse RNA polymerase I. We are also grateful to M. Hohloch for preparing the photographs. We thank Dr. T. Focareta, Tiibingen, for reading the manuscript. H.V. is the recipient of a research fellowship from the Graduiertenf~rderung des Landes BadenWiirttemberg. This work was supported by the Deutsche Forschungsgemeinschaft (Bu 483).
References 1 Hausen, P. and Stein, H. (1970) Eur. J. Biochem.14, 278-283. 2 Crouch, R.J. and Dirksen, M.-L. (1982) Cold Spring Harbour Monographs, Vol. 14, 211-241. 3 Btisen,W. and Hausen, P. (1975) Eur. J. Biochem.52, 179-190. 4 Biisen, W. (1976) Dissertation, Universit~itTiibingen. 5 Vonwirth, H. (1987) Diplomarbeit, Universit~itTiibingen. 6 Vonwirth, H., Frank, P. and Biisen, W. (1989) Eur. J. Biochem. 184, 321-329. 7 Vonwirth, H., Frank, P. and Biisen, W. (1990) Experientia 46, 319-321. 8 Btisen, W. (1980) J. Biol. Chem. 255, 9434-9443. 9 Biisen,W. (1982) J. Biol. Chem. 257, 7106-7108. 10 Btisen, W. (1980) in Biological Implications of Protein-Nucleic Acid-lnteractions (Augustyniak, J., ed.), pp. 571-584, Elsevier/ North-Holland, Amsterdam.
38 11 Biisen, W., Peters, J.H., Hausen, P. and Crystalla, G. (1977) Eur. J. Biochem. 74, 203-208. 12 Tsukada, K., Sawai, Y., Saito, I. and Sato, F. (1978) Bioch. Biophys. Res. Commun. 85, 280-286. 13 Sawai, Y., Kitahara, N., Thung, W.L., Yanokura, M. and Tsukada, K. (1981) J. Biochem. 90, 11-16. 14 Huet, J., Wyers, F., Buhler, J.-M., Sentenac, A. and Fromagoet, P. (1976) Nature 261, 431-433. 15 Huet, J., Buhler, J.-M., Sentenac, A. and Fromagoet, P. (1977) J. Biol. Chem. 252, 8848-8855. 16 lborra, F., Huet, J., Breant, B., Sentenac, A. and Fromagoet, P. (1979) J. Biol. Chem. 254, 10920-10924. 17 Stein, H. and Hausen, P. (1970) Eur. J. Biochem. 14, 270-277. 18 Dahmus, M.E. (1981) J. Biol. Chem. 256, 3332-3339. 19 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 20 Laemmli, U.K. (1970) Nature 227, 680-685. 21 Oakley, B.R., Kirsch, D.R. and Morris, N.R. (1980) Anal. Biochem. 105, 361-363. 22 Merril, D., Goldman, D. and Van Keuren, M.L. (1984) Methods Enzymol. 104, 441-447. 23 Dignam, J.D., Lebovitz, R.M. and Roeder, R.G. (1983) Nucleic Acids Res. 11, 1475-1489.
24 Dulbecco, R. and Vogt, M. (1954) J. Exp. Med. 99, 167-182. 25 Gissinger, F. and Chambon, P. (1972) Eur. J. Biochem. 28, 277282. 26 Kedinger, C., Gissinger, F. and Chambon, P. (1974) Eur. J. Biochem. 44, 421-436. 27 Sentenac, A. (1985) CRC Crit. Rev. Biochem. 18, 31-90. 28 Tower, I. and Sollner-Webb, B. (1987) Cell 50, 873-883. 29 Stetler, D.A., Rose, K.M., Wenger, M.E., Berlin, C.M. and Jacob, S.T. (1982) Proc. Natl. Acad. Sci. USA 79, 7499-7503. 30 Karwan, R. and Kindas-Mtigge, I. (1989) Eur. J. Biochem. 179, 549-555. 31 Wintersberger, U., Kiihne, C. and Karwan, R. (1988) Biochim. Biophys. Acta 951, 322-329. 32 Vonwirth, H. (1990) Dissertation, Universit~it Tiibingen. 33 Kane, C.M. (1988) Biochemistry 27, 3187-3196. 34 Roberge, M. and Bradbury, E.M. (1988) J. Biol. Chem. 263, 18553-18557. 35 Grummt, I. (1981) Proc. Natl. Acad. Sci. USA 78, 727-731. 36 Kuhn, A. and Grummt, I. (1989) Genes Dev. 3, 224-231.
31
Elsevier BBAEXP 92156
Class II ribonuclease H comigrates with, but is distinct from, the third largest subunit of calf thymus RNA polymerase I Heiner Vonwirth
1, P e t e r
Frank
1, C l a u d e
Kedinger 2 and Werner Biisen 1
1 Lehrstuhlfiir Allgemeine Genetik, Biologisches Institut, Tiibingen (F.R.G.) and e Laboratoire de Genetique Moleculaire des Eucaryotes du CNRS, Strasbourg (France)
(Received 6 February1990)
Key words: RibonucleaseH; RNA polymeraseI; Autoimmuneantibody It has been reported (Iborra et al. (1979) J. Biol. Chem. 254, 10920-10924) that the third and the fifth largest subunit of yeast RNA polymerase I exhibit ribonuclease H activity. The authors suggested that the third largest subunit is identical with the chromatin-associated ribonuclease H49 , the putative yeast equivalent of bovine ribonuclease H lib. Although the third largest subunit of calf thymus RNA polymerase I and ribonuclease H Ilb display nearly identical molecular masses under denaturing conditions, serological analysis reveals that, in contrast to their counterparts in yeast, these mammalian proteins are distinct entities. Interestingly, sera from some patients with mixed connective tissue disease which contain antibodies directed against RNA polymerase I, also react with ribonuclease H Ilb epitopes. This observation suggests that a protein displaying ribonuclease H lib antigenicity could be associated with RNA polymerase I. Additional indications supporting this conclusion are discussed.
Introduction Enzymes with the specificity of a ribonuclease H (EC 3.1.13.2) first discovered by Hausen and Stein [1], are widespread among pro- and eukaryotes (for a review see Ref. 2). In calf thymus, three distinct ribonuclease H activities, named H I, H IIb and H IIa, are found. Whereas H I and H IIb display ribonuclease H specificity, H IIa lacks this specificity [3-7]. Enzymes I and IIb were purified and polyclonal antibodies were raised against these protein fractions. Antibodies directed against ribonuclease H I inhibit only this enzyme activity [8,9]. Antibodies raised against ribonuclease H IIb deplete enzyme extracts from the antigenic protein band and enzyme activity [6]. Both enzymes cannot only be differentiated at the serological level but also by their physical and biochemical parameters, including their mode of action [3-10]. Ribonuclease H I, an endonuclease, and H IIb, presumably an exonuclease, seem
Abbreviations: SDS, sodium dodecyl sulfate; PMSF, Phenylmethylsulfonyl fluoride; DTr, Dithiothreitol; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethane sulfonicacid; MCTD, mixed connectivetissue disease. Correspondence: W. Btisen, Lehrstuhl fiir AllgemeineGenetik, Bioiogisches Institut, Universit~it Ttibingen, Auf der Morgenstelle 28, D-7400 Tiibingen,F.R.G.
to play different functional roles in vivo, as suggested by observations that their activity levels are elevated at different times after the stimulation of resting bovine lymph node cells with concanavalin A. Ribonuclease H IIb activity parallels the increase in RNA synthesis, whereas ribonuclease H I increases concomitant with the rise in DNA synthesis [11]. Further indications for a relationship between the class II ribonuclease H and transcription have been reported in the literature. Tsukada et al. [12] and Sawai et al. [13] found that the liver nuclei of thioacetamide treated rats show a parallel increase of RNA polymerase I (EC 2.7.7.6) and class II ribonuclease H activity. The class I ribonuclease H and RNA polymerase II activity remained nearly unaffected by this treatment. This does not exclude a possible link between the other RNA polymerase species (II and III) and class II ribonuclease H, but it stresses the interaction between R N A polymerase I and class II ribonuclease H. Further support for this link was derived from the findings of Iborra et al. [14-16] who reported that the third and the fifth largest subunit of RNA polymerase A (I) of yeast possesses ribonuclease H activity. This association has not been confirmed in yeast so far, nor has it been shown to exist in higher eukaryotes. Because of the importance of this finding and the possible functional implications, we have asked whether RNA polymerase I, and in particular the third largest
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)
32 subunit of calf thymus RNA polymerase I displays ribonuclease H lib antigenicity. Here we report, that the molecular masses of the ribonucleases H lib were comparable to those reported for the third largest subunits of the corresponding RNA polymerases I in calf thymus and mouse cells. In more or less purified calf thymus samples we found a protein with ribonuclease H lib antigenicity coeluting with RNA polymerase I. However, serological analysis revealed that the calf thymus protein displaying ribonuclease H lib antigenicity and the third largest subunit of calf thymus RNA polymerase I were distinct from each other. We discuss indications for a possible specific binding of class II ribonuclease H to RNA polymerase I in mammalian systems. Materials and Methods
Assay for ribonuclease H. The standard [3H]RNA. DNA hybrid was prepared as described in previous communications [3]. The specific activity of the hybrid was 66 d p m / p m o l ribonucleotide. Ribonuclease H lib assays were carried out as described elsewhere [3,6]. Assay for RNA polymerase. RNA polymerase activity was measured according to Stein and Hausen, and Dahmus [17,18] with minor modifications. A final assay volume of 100 #1 containing 30 mM Tris-HC1 (pH 7.8), 50 mM (NH4)2SO4, 2 mM MnC1 z, 1 mM dithiotreitol (DTT), 1 mM each of ATP, CTP and GTP, 0.1 mM [3H]UTP (400 d p m / p m o l ) , 12.5 /xg of non-denatured calf thymus DNA, and various amounts of enzyme solution was incubated for 10 rain at 37 ° C. The reaction was stopped by addition of 100 /~1 8% trichloroacetic acid and incubation for 10 rain on ice. The resulting precipitate was collected on a Na4P20 7saturated nitrocellulose filter (Millipore), washed, dried, and counted in a liquid scintillation counter. 200 cpm correspond to 1 pmol ribonucleotides incorporated. Protein determination. Protein concentration was determined according to Bradford using bovine serum albumin as a standard [19]. SDS-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in the presence of SDS was carried out in 1-nun-thick slab gels according to Laemmli [20]. After electrophoresis, the gels were processed for silver staining [21,22]. Molecular masses were determined as described [6]; myosin (205 kDa) was used as an additional marker. Immunological detection of proteins. Western blotting experiments were carried out as described [6]. The nitrocellulose sheets were blocked with bovine serum albumin and incubated overnight with rabbit serum (either control- or antiserum) or human-autoimmune serum as indicated in the individual experiments. Binding of IgG was visualized by incubation with peroxidase-conjugated goat-anti-rabbit or goat-anti-human IgG (H +
L) followed by peroxidase reaction.
Preparation of monospecific antibodies. Antibodies bound to distinct protein bands were eluted and used directly for immunological analysis as described previously [6]. Preparation of nuclear extracts. Nuclear extracts from mouse Ehrlich Ascites and HeLa cells were prepared according to D i g - a m et al. [23]. Cells were grown to a density of about 6 . 1 0 5 cells per ml. The cells were harvested from the culture medium by centrifugation for 10 min at 200 x g and 4°C. The cell pellet was washed twice with cold N a C 1 / P i (phosphate-buffered saline according to Dulbecco [24], but without Ca 2+ and MgZ+), and suspended in five packed-cell volumes of buffer A (10 mM Hepes (pH 7.9), 1.5 mM MgC12, 10 mM KC1 and 0.5 mM DTT) and allowed to stand for 10 min at 4 ° C . The suspension was centrifuged for 10 min at 200 X g and 4 ° C and the pellet was resuspended in two packed-cell volumes of Buffer A. The cells were lysed by 15 strokes with a Dounce homogenizer and centrifuged for 10 min at 200 x g and 4 o C. The supernatant was decanted and processed for preparation of the S100 fraction exactly as described [23]. The pellet was again centrifuged (20 min at 25 000 x g and 4 ° C) and the sediment of the second centrifugation step suspended in buffer C (20 mM Hepes (pH 7.9), 25% glycerol, 0.42 M NaC1, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT) (3 ml/109 cells) using a Dounce homogenizer. The suspension was stirred gently for 30 min at 4 ° C and centrifuged for 30 min at 2 5 0 0 0 x g and 4 ° C . The supernatant was dialysed against 50 volumes of buffer D (20 mM Hepes (pH 7.9), 20% glycerol, 0.1 M KC1, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT). The dialysate was centrifuged as above and the clear supernatant frozen as aliquots in liquid nitrogen and stored at - 7 0 ° C. The protein concentrations of the S100 fractions were between 2 and 8 m g / m l and those of the nuclear extracts between 5 and 10.4 mg/ml. Anti-calf thymus ribonuclease H lib-affinity chromatography. The anti-calf thymus Ribonuclease H IIb affinity matrix was prepared as described [6]. The enzyme solution was incubated overnight at 4 ° C with the affinity matrix. The suspension was filtered through a Btichner funnel and the flow through collected. Ribonuclease H activity was determined in the enzyme solution before adsorption to the matrix and in the flowthrough fraction.
Preparation of calf thymus crude extracts and separation of RNA polymerases. Calf Thymus crude extracts were prepared as described previously [6]. The crude extract from 75 g frozen calf thymus was diluted 10-fold with buffer B (30 mM Tris/HC1 (pH 7.8), 2 mM EDTA, 0.1% 2-mercaptoethanol (v/v), 30% glycerol (w/v)) and adsorbed onto DEAE-cellulose (140 g of preswollen DE52, Whatman). The matrix was washed
33 extensively with buffer B, loaded onto a column (4.9 cm2 × 40 cm), and the proteins were eluted with a linear salt gradient (0-1000 mM KC1 in buffer B). Fractions of 2 ml were collected and the RNA polymerase activity of 20 gl of the individual fractions was determined, without and with the addition of a-amanitin (2 # g / m l assay). Under these conditions RNA polymerase II activity is strictly inhibited while RNA polymerase I activity is not affected. Calf thymus RNA polymerase L" enzyme and specific antiserum preparation. Calf thymus RNA polymerase I (originally named AI) was purified to apparent homogeneity, as described previously [25]. The experiments reported hereafter were performed with the glycerolgradient fraction (GG) concentrated to 10 mg protein per ml by vacuum-concentration through a Sartorius dialysis bag. Antibodies were raised against an independent preparation of RNA polymerase I fraction GG [26] which gave a SDS-polyacrylamide gel electrophoresis pattern indistinguishable from that of the fraction used in the immunoblot analysis. Calf thymus ribonuclease H IIb: enzyme and specific antiserum preparation. Calf thymus ribonuclease H IIb was purified to apparent homogeneity as described previously [6]. The enzyme fraction used in this report corresponds to the most purified enzyme fraction (fraction 7) [6]. Antibodies were those used in the previous study [6].
A utoimmune sera. Sera used were from clinically identified mixed connective tissue disease (MCTD) patients. They were also shown to contain antibodies directed against U1-RNP (R. Liihrmann, Marburg, personal communication). Chemicals and isotopes. All chemicals used were reagent grade and obtained from Boehringer Mannheim, Merck, Serva and Sigma. [3H]UTP was from Amersham International (U.K.). Ribo- and deoxyribonucleoside triphosphates, native calf thymus DNA, protein markers, E. coli RNA polymerase, yeast RNA and PMSF were obtained from Boehringer (Mannheim, F.R.G.). Results
Correspondence of the molecular masses of ribonuclease H lib and the third largest subunit of RNA polymerase I in mammalia Although the biological function of a eukaryotic class II ribonuclease H is not known, several lines of evidence point to a function during the transcription process, as indicated in the introduction. Unfortunately these findings, particularly the association of ribonuclease H and RNA polymerase I, have been neither confirmed nor disproven in yeast or in any other eukaryotic system. So far we have failed to find ribonuclease H enzymatic activity in association with mammalian RNA
HeLa A
A
C
Calf Thymus C
116-94-67--
6!6-o52-45. 40--
25--
1 2
3
1
2
3
~
2'
3'
1'
2'
3'
Fig. 1. Mammalian ribonuclease H Ilb-antigenicity is associated with two fractions. HeLa cell nuclear extracts and calf thymus crude extracts were adsorbed to DEAE-CeUulose and the 200 mM KCI-DEAE-ceUulose eiuates, together with the DEAE-cellulose flow-through fractions, were analysed by immunoblotting using the antiserum directed against calf thymus ribonuclease H IIb (A) and the control serum (C). Proteins were separated on 12% polyacrylamide gels and processed for immunoblotting as described in Materials and Methods. Antiserum (A) was diluted 1:1000, control serum (C) 1:500. Lanes 1, 2 and 3 correspond to 25 gg of protein of the HeLa cell nuclear extract, of the 200 mM KC1-DEAE-cellulose eluate, and of the DEAE-cellulose flow-through, respectively. Lanes 1', 2' and 3' correspond to 10 #g of protein of calf thymus crude extract, to 100 #g of protein of the 200 mM KC1-DEAE-cellulose eluate, and to 10 #g of protein of the DEAE-cellulose flow-through. The slightly different migration behavior of the antigenic band in lane 3' in comparison to that in lane 2' is due to band curvature at the margin of the gel.
34 polymerase I, but the availability of specific antisera against calf thymus ribonuclease H lib and RNA polymerase I, and of highly purified protein fractions of both enzymes allowed us to explore the situation in mammalian systems at the serological level. In a previous study we identified calf thymus ribonuclease H IIb as most likely a monomeric enzyme with apparent molecular masses of 45 and 52 kDa under native and denaturing conditions, respectively [6]. Interestingly, the 52 kDa value corresponds very well to the 51 kDa reported for the third largest subunit of calf thymus RNA polymerase I [26]. To investigate whether this correlation also holds true for other mammalian systems, proteins of HeLa cell and mouse nuclear extracts were processed for western blotting. Fig. 1 (lane 1, A) shows that the anti-calf thymus ribonuclease H IIb-serum cross-reacted specifically and predominantly with a 61 kDa protein band but also with a minor 52 kDa protein band of the HeLa cell nuclear extract. This finding has been confirmed by immunoblots using a monospecific antibody isolated from affinity-purified calf thymus ribonuclease H lib (see Materials and Methods) (not shown, Ref. 32). Depletion experiments with fractions containing exclusively ribonuclease H lib activity (see Materials and Methods) revealed that the antigenic protein bands are quantitatively and the enzyme activity is almost quantitatively retained by the anti-calf thymus ribonuclease H lib-affinity matrix (not shown, Ref. 32). From these data we conclude that ribonuclease H IIb activity must be associated with the 61 and 52 kDa-proteins. Gel filtration experiments revealed a native molecular mass of 45 kDa (not shown, Ref. 32), the same value which was determined for the calf thymus enzyme [6]. We therefore suggest that this HeLa enzyme activity is the human homologue to calf thymus ribonuclease H lib [32]. A ribonuclease H with properties
l 3.Mr
similar to our calf thymus ribonuclease H IIb was recently isolated from HeLa cells [33]. The final preparation was not homogeneous, but it is striking to see that the two most prominent protein bands of the most purified fraction show molecular masses between 50 and 60 kDa. There is no report in the literature on the molecular mass of the third largest subunit of human RNA polymerase I, but it is interesting to note that the molecular mass for the third largest subunit of mouse RNA polymerase I has been reported to be 61 kDa under denaturing conditions [27]. Experiments performed with mouse nuclear extracts also revealed a 61 kDa-protein to be crossreactive (not shown, Ref. 32). These initial, preliminary results were compatible with the idea that the third largest subunit of mammalian RNA polymerase I is ribonuclease H lib.
Less purified fractions of RNA polymerase I display ribonuclease H lib antigenicity DEAE-cellulose chromatography separates ribonuclease H lib and RNA polymerase I activities from each other (the former one stays in the flow-through fraction whereas the latter one is retained on this matrix). If ribonuclease H IIb is identical with the third largest subunit of RNA polymerase I, the antibody directed against calf thymus ribonuclease H lib should reveal antigenicity not only to proteins present in the flow-through, but also to proteins bound to the matrix. To address this question, proteins of the crude extract (Fig. 1 lanes 1, 1'), the DEAE-cellulose flow-through (lanes 3, 3'), and the fraction bound to the matrix (lanes 2, 2') were processed for immunoblotting. As can be seen in Fig. 1, all fractions of HeLa cells and calf thymus tissue displayed ribonuclease H lib antigenicity in association with proteins of the expected size. According to the protein recovery from the DEAE-cel-
0,0 0,05 0,1 0,2 0,5 1,0 0,7 0,5 0,45 0/,5 0,35
RNA Pol. I Activity
116-94-67-60-40--
25--
33
35 3"7 39
M
43 45 47 49 51
53
Fraction Number
Fig. 2. A protein with fibonucle~e H l i b anfigenicity coelutes on DEAE-ceUulose with R N A polymer~se ! of calf thymus. C~lf thymus crude
extract was adsorbed to DEAE-cellulose,the proteins were eluted with a linear salt gradient and the individual fractions were tested for RNA polymerase activity. Proteins of the indicated fractionswere separated on a 12% polyacrylamidegel and processed for immunoblottingusing the anti-ribonucleaseH lib-serum. The a-amanitininsensitiveRNA polymeraseactivityis indicated in relativeunits along the top of the figure.
35 lulose chromatography (not shown), the intensities of the antigenic protein bands suggest a more or less equal distribution of the antigens between the fraction binding to DEAE-cellulose and the flow-through fraction in HeLa cells, and only about 5% antigenicity associated with the DEAE-cellulose-retained fraction from calf thymus (under the conditions used, the DEAE-cellulose flow-through fraction was quantitatively depleted from ribonuclease H I, [3,8] (not shown)). This result is remarkable because ribonuclease H lib enzymatic activity is found only in the DEAE-cellulose flow-through fraction [3,6]. In a next step we wanted to see whether the proteins binding to DEAE-cellulose and displaying ribonuclease H lib antigenicity coeluted with RNA polymerase I activity. We adsorbed calf thymus crude extract to DEAE-cellulose, eluted the bound proteins with a linear salt gradient, and tested the gradient fractions for RNA polymerase activity as described in Materials and Methods. We identified the fractions containing RNA polymerase I and II activity and tested these fractions for crossreaction with the polyclonal antibody against ribonuclease H lib by immunoblotting. Fig. 2 shows an immunoblot throughout the (a-amanidn insensitive) RNA polymerase I activity peak. As can be seen, antigenicity coelutes with the beginning and the left half of the RNA polymerase I activity peak. Under the assumption that no inhibitors interfere with RNA polymerase activity and that the measurable RNA polymerase activity reflects the distribution of RNA polymerase molecules, one could state that ribonuclease H IIb antigenicity is restricted to a subfraction of RNA polymerase I. Indications for more than one form of RNA polymerase I are reported in the literature [27,28]. Antibodies directed against calf thymus RNA polymerase I recognize ribonuclease H lib To further prove the postulated association of ribonuclease H IIb and RNA polymerase I, we performed immunoblots with fractions of several different purification steps of calf thymus ribonuclease H lib and polyclonal antibodies directed against this enzyme ( a l l IIb) and calf thymus RNA polymerase I (aPol I). Fig. 3 shows an example of such an experiment performed with the most purified ribonuclease H lib (fraction 7, Ref. 6). A strong antigenic response to the 52 kDa ribonuclease H lib protein was detected with antiserum directed against ribonuclease H lib, as well as with antiserum raised against RNA polymerase I. The specificity of this reaction is underlined by the fact that only about 5% of the polypeptides comprised by the most purified ribonuclease H lib fraction have a molecular mass of 52 kDa under denaturing conditions (Fig. 3, lane t). The remaining peptides of lower molecular masses correspond to degradation products of the native enzyme [6]. Thus, both sera react specifically with
lO~.Mr ~HIIb . P o l I C
116
t ..........
9Z,~ 67~
60
524~-:_ 40--
-
25--
Fig. 3. Immunoblot analysis of purified calf thymus ribonuclease H lib. Purified calf thymus ribonuclease H IIb (4 #g of protein of fraction 7, Ref. 6) was separated and processed for immunoblotting. aH lib (anti-ribonuclease H lib serum, 1:1000), ttPol I (anti-RNA polymerase I-serum, 1:1000), C (control serum, 1:500), t (silver staining of 4/~g of purified ribonuclease H IIb).
the intact 52 kDa ribonuclease H IIb polypeptide chain. We conclude that even the highly purified calf thymus RNA polymerase I fraction used to raise antibodies must have contained ribonuclease H lib antigenic proteins. Human autoimmune sera react specifically with ribonuclease H lib It is known from the literature that sera from patients with the mixed connective tissue disease (MCTD) detect RNA polymerase I specifically, and not the RNA polymerases II and III. Immunoblotting analysis has shown that some of these sera contain antibodies directed against the third largest subunit of RNA polymerase I [29]. Thus, if there is an identity of this subunit with the ribonuclease H lib protein, highly purified ribonuclease H lib should be also recognized by some sera from MCTD patients. We examined 14 different sera from MCTD-patients and looked for crossreaction of these sera with highly purified calf thymus ribonuclease H IIb. In 7 out of 14 sera we found a clear crossreaction with the ribonuclease H lib-specific 52 kDa-protein band. Fig. 4 shows immunoblots performed with two of the cross-reacting autoimmune sera. Strong crossreactivity was found in association with either the 52 kDa protein band alone (left) or the 52 and the 45 kDa protein bands (right). These data suggest that ribonuclease H lib serves as an antigen in at least some MCTD-patients. The third largest subunit of RNA polymerase I is distinct from ribonuclease H IIb The data presented above do not discriminate between the possibilities that the third largest subunit of
36 RNA polymerase I and ribonuclease H IIb are associated with one and the same polypeptide chain, that ribonuclease H IIb is specifically associated with RNA polymerase I but distinct from the third largest subunit, or that ribonuclease H lib simply copurifies with RNA polymerase I. Highly purified samples of calf thymus RNA polymerase I made a decision possible. Fig. 5 presents immunoblots performed with highly purified calf thymus RNA polymerase I (fraction GG, see Fig. 5 of Ref. 25 and Fig. 2 of Ref. 26; lane 1) and highly purified calf thymus ribonuclease H lib (fraction 7, Ref. 6; lane 2). As can be seen in lane 1, aPol I, the RNA pol I antiserum detected the four largest RNA polymerase I subunits with molecular masses of 205, 127, 53, and 43 kDa under our conditions. On the contrary, the ribonuclease H lib antiserum did not recognize any of the RNA pol I subunits (lane 1, aH IIb). This finding proves that the third largest subunit of RNA polymerase I and ribonuclease H lib are distinct from each other. At the first glance this seems to be a contradiction to the result presented in Fig. 3, in which it was shown that the RNA pol I antiserum cross-reacted with the most purified ribonuclease H IIb fraction. To resolve this question we isolated, from the RNA pol I antiserum, monospecific antibodies directed against the ribonuclease H IIb specific 52 kDa protein band (aPol Im52). AS can be seen in Fig. 5 lanes 1 and 2, aPol Im52, this subfraction of the RNA pol I serum does recognize the ribonuclease H IIb specific 52 kDa protein band but neither the third largest subunit of RNA polymerase I nor any other subunit. For that reason we state that the
MCTD-Sera 116-94-67--
526043-= 40--
25--
Fig. 4. Immunoblot analysis of purified calf thymus ribonuclease H lib by use of autoimmune sera. Purified calf thymus ribonuclease H IIb (4 /~g of protein, see Fig. 3) was separated and processed for immunoblotting. MCTD-sera were diluted 200-fold.
103Mr
,PolI ,tH2~b C
205--
.~HIIb
,,Po/ Im52
m
116--
67-GO--
45--
I
I
1
I
2
I
2
Fig. 5. Immunoblot analysis of purified calf thymus RNA polymerase I and calf thymus ribonuclease H lib with distinct antisera. Purified calf thymus RNA polymerase I (12 #g of protein) (lanes 1) and purified calf thymus ribonuclease H lib (4 #g of protein, see Fig, 3) (lanes 2) were separated on 8% SDS-polyacrylamide gels and processed for immunoblottings, aPol I (anti-RNA polymerase l-serum, 1 : 1000), aH IIb (anti-ribonuclease H IIb-serum, 1:1000), C (control serum, 1 : 500), aPol Ira52 (monospecific antibodies isolated out of the polyclonal anti-RNA polymerase I-serum after binding to the affinity purified ribonuclease H lib specific protein band with a molecular mass of 52 kDa).
purified RNA polymerase I fraction used to raise antibodies must have contained a protein with ribonuclease H IIb antigenicity. The highly purified RNA polymerase I fraction used in the experiments shown in Fig. 5 must have lost this protein during the purification procedure. These results indicate that, depending on the preparation, highly purified RNA polymerase I can contain an additional protein with ribonuclease H IIb antigenicity, and that the third largest subunit of RNA polymerase I is distinct from the protein with ribonuclease H lib antigenicity. Discussion
More than 10 years ago Huet et al. [14,15] showed that highly purified yeast RNA polymerase I displays ribonuclease H activity. Moreover, Iborra et al. [16] were able to show that the third largest subunit of RNA polymerase I is identical with the chromatin associated ribonuclease H49. These results, together with the data presented by Biisen et al., Tsukada et al., and Sawai et al. (see Introduction), which showed that class II ribonuclease H activity is regulated with transcription and RNA polymerase I activity, respectively (Refs. 11-13), are in favor of the interpretation that the association
37 found by Huet et al. [14,15] is biologically relevant. It is therefore more than surprising that these interesting results were neither confirmed nor disproven in yeast or in any other eukaryotic system. In this paper we could show, by using different types of antibodies specific for either RNA polymerase I or ribonuclease H IIb, that in calf thymus the third largest subunit of RNA polymerase I and ribonuclease H lib correspond to two distinct polypeptide chains. This is in variance to what has been reported for yeast RNA polymerase I. Nevertheless, proteins showing ribonuclease H IIb antigenicity can be found in highly purified fractions of RNA polymerase I. This could mean that RNA polymerase I and ribonuclease H lib bind specifically to each other and that both proteins may copurify through several purification steps. This conclusion is supported by the following observations: (1) Whereas ribonuclease H lib does not bind to DEAE-cellulose, a protein with ribonuclease H lib antigenicity coelutes with RNA polymerase I from this matrix. (2) The findings that all sera of MCTD patients recognize RNA polymerase I specifically [29] and that some MCTD-sera - as shown in this report - recognize ribonuclease H lib in addition, are compatible with the notion that some patients develop sera against a RNA polymerase I-ribonuclease H IIb complex. In this context it should be mentioned that anti-Sm autoantibodies, prototype antibodies reacting with antigens of RNA-protein complexes and often produced by MCTD-patients, are reported to detect specifically yeast ribonuclease H55 [30], the enzyme of a proteinase-deficient yeast strain, believed to correspond to yeast ribonuclease H49, characterized by Iborra et al. [16,31]. (3) Recently, we separated at least two forms of RNA polymerase I in the protozoan Crithidia fasciculata, from which only one displays ribonuclease H IIb antigenicity. (4) Coelution of this antigen with RNA polymerase I can be followed through the whole purification procedure, including gel filtration under 0.1 M (NH4)2SO 4 (Ref. 32; K~Sck, J. et al., unpublished data; Vonwirth, H. et al., unpublished data). Roberge and Bradbury studied the interactions between nascent R N A and RNA polymerase I-transcribing complexes in HeLa cell nucleoli by photoaffinity labeling [34]. They observed a specific labeling of the two largest subunits of RNA polymerase I and of a 52 kDa protein band. In the light of the data presented by Huet et al. [14,15] and Iborra et al. [16] they speculate that the photoaffinity-labeled 52 kDa protein band is the human equivalent of the yeast third largest subunit of RNA polymerase I and therefore also of the yeast ribonuclease H. As an alternative, they propose that the 52 kDa protein band is not a subunit of RNA polymerase I but a distinct polypeptide intimately bound to the transcribing polymerase. Our data do not support
their first suggestion. However, our results do not allow to differentiate whether the 52 kDa protein band is the third largest subunit of HeLa cell RNA polymerase I or the human equivalent of our protein displaying ribonuclease-H lib antigenicity or another unknown protein. All data presented in this paper and others (see Introduction) render unlikely a fortuitous association of ribonuclease H activity a n d / o r ribonuclease H IIb antigenicity with RNA polymerase I. Nevertheless, final proof has to await for functional correlations during the RNA polymerase I transcription process. Since ribonuclease H activity could not be demonstrated for the protein displaying ribonuclease H lib antigenicity, we have to be also aware of the possibility that an enzymatic activity different from ribonuclease H activity is important for its function in vivo. Answers can be expected from studies using highly purified ribonuclease H lib, RNA polymerase I with and without the associated protein with ribonuclease H IIb-antigenicity and the specific antibody against ribonuclease H IIb in in vitro systems for proper initiation or termination of RNA polymerase I transcription [35,36].
Acknowledgements The advice and support of Prof. Dr. W. Seyffert is gratefully acknowledged. We are indebted to Prof. Dr. R. Liihrmann (Marburg) for kindly supplying us with the diverse sera of MCTD patients, for critically reading the manuscript and for helpful advice. Thanks also to Prof. I. Grummt (Wtirzburg) for the kind gifts of nuclear extracts of mouse Ehrlich ascites cells and purified fractions of mouse RNA polymerase I. We are also grateful to M. Hohloch for preparing the photographs. We thank Dr. T. Focareta, Tiibingen, for reading the manuscript. H.V. is the recipient of a research fellowship from the Graduiertenf~rderung des Landes BadenWiirttemberg. This work was supported by the Deutsche Forschungsgemeinschaft (Bu 483).
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