Characterization and subcellular localization of ribonuclease H ...

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33076 Bordeaux Cedex, France and the lEehrstuhl fur Allgemeine Genetik, Biologisches ...... Henry, C. M., Ferdinand, F. J., and Knippers, R. (1973) Biochem.
Vol. 269, No. 40, Issue of October 7,pp. 25185-25192, 1994 Printed in U.S.A.

Cmanm~ b l JOURNAL OF BIOL~CICAL 0 1994 by The American Society for Biochemistry and Molecular Bioloa, Inc

Characterization and Subcellular Localizationof Ribonuclease H Activities from Xenopus Zaevis Oocytes* (Received forpublication, April 7, 1994, and in revised form, July 27, 1994)

Christian Cazenave$P,Peter Frankflll, Jean-Jacques ToulmeS, and Werner Busenn From the +L,aboratoire de Biophysique Moleculaire, INSERM U386, Universite de Bordeaux ZI, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France and the lEehrstuhl fur Allgemeine Genetik, Biologisches Znstitut, Uniuersitat Tubingen, Auf der Morgenstelle 28, 72076 Tiibingen, Germany

Ribonuclease H activities present in fully grown Xe- known in great detail (16, 17).Similarly, numerous genetic nopus oocytes were investigated by using either liquid studies performed mostly with E. coli have demonstrated the assays or renaturation gel assays. Whereas the test in role of RNase HI in the precise initiation of DNA replication of solution detected an apparently unique class I ribonu- the bacterial chromosome (18)or plasmids (19); moreover the clease H activity, the activity gels did not detect this crystal en- structureof this enzyme has been recently determined zyme but another one with the molecular weight ex- (20, 21). Although the detailed structure and function of the I1 ribonuclease H. The ribonucleaseHI eukaryotic enzymes is unknown, a generalpicturecan pected for a class be was found to be primarily concentrated in the germinal drawn from the various attempts to characterize these envesicle, but around 5%of this activity was detected in the zymes further: allcells from lower and higher eukaryotes so far cytoplasm and may correspond to the activity involved in known t o contain at least two forms of RNase H tested are antisense oligonucleotide-mediated destruction of mes- activity, a class I and a class I1 ribonuclease H (22). These senger RNAs. The concentration of this class I ribonuenzymes can be differentiated by physical, biochemical, and clease H in oocytesis similar to that in somatic cells. The believed to function during class I1 ribonuclease H remained undetectable by the test serological parameters and they are in solution becauseits activity was cryptic.On activity DNA replicationand RNA transcription, respectively (23). These enzymesplay a critical role in thesuccess of the so-called gel, a polypeptide with the apparent molecular of 32 mass olim a , expected for a ribonuclease HII, was found to be “hybrid arrest of translation” experiments using antisense H ac- godeoxynucleotides (24). This has been shown in cell-free exconcentrated in mitochondria although no RNase tivity could be detected by using the liquid assay. Basedtracts. Xenopus oocytes are theonly cells in which the involveeffect has beenclearly ment of RNase H inantisense on sedimentation studies, we hypothesize that the apparestablished (25, 26). Indeed, chemically modified oligos which ent absence of RNaseH activity in solution could be the result of the associationof this 32-kDa polypeptide with do not allow RNase H activity fail to inhibit translation unless other polypeptides, or possibly nucleic acids, to forma the cap site or the initiationcodon region are targeted(27,281. multimer of, until now,unknown function. Interestingly, the presence of RNase H activity in the cytoplasm ofXenopus oocytes has been indirectly proved: antisense oligonucleotides induced the selective degradation of mature Ribonucleases H (RNases H for short) are RNA-degrading mRNAs injected in the cytoplasm (25). The cytoplasmic localenzymes active only on RNA hybridized to a complementary ization of this activity had tobe exploredfurther as this finding DNA strand. This activityhas been first identified in calf thy- was rather unexpected in view of the putative functions of mus (1, 2) and soon afterwards in a retrovirus in association eukaryotic RNases H in replication (231, transcription (231, or DNA repair (29, 30) which would have rather argued for a (3). Thereaftersimilaractivities withreversetranscriptase were recognized in Escherichia coli cells (4-6) and in a great nuclear localization. As this cell is arrested inmeiotic prophase cytoplasm someproteins which will number of eukaryotic cells of various origins, as for example, and isknown t o store in the yeast (71, rat liver (81, and rat brain (91, tumor cells (10,111, and migrate t o the nuclei later during embryonic development it H belongs to this plant cells (12) (for reviews, see Refs. 13 and 14 and Crouch and was of interest to investigate whether RNase set of maternal proteins. Toulme (64)). In an attempt to obtain a better knowledge about the parDespite the fact that these enzymes have been first found more than 20 years ago in cells of higher eukaryotes theirexact ticipation of RNases H in antisense-mediated arrest of transunknown. In lation, we started investigating the nature and subcellularlofunction in nucleic acidmetabolism is still largely Xenopus oocytes. contrast, a much better knowledge of the structure and fmc- calization of the RNase H activities present in tion of the RNase H associated with reverse transcriptase has MATERIALS AND METHODS accumulated in the last years (15) and the role of RNase H Xenopus Oocytes-Xenopus laeuis frogs (purchased from Centre de activity in the whole process of reverse transcription is now Recherches de Biochimie Macromoleculaire, CNRSin Montpellier)were anesthesized with1g/liter MS222 (Sandoz)and their ovaries surgically * The costsof publication of this article were defrayed in part by the removed, dilacerated in modified Barth’s saline (MBS)’ (31),and the payment of page charges. This article must therefore be hereby marked oocytes defolliculated at room temperature under constant gentle stir“aduertisement”in accordance with 18 U.S.C. Section 1734 solely to ring by treatment with 0.4mglml dispase in MBS for 4 h, then 1mg/ml indicate this fact. collagenase in MBS for 1 h. Thereafter the oocytes were extensively $ Charge de Recherches at CNRS. To whom correspondence should be washed with MBS. Fully grown stage VI oocytes (32) were manually addressed: Laboratoire de Biophysique MolBculaire, INSERM U386, Universite de Bordeaux 11,146rue Leo Saignat,33076 Bordeaux Cedex, selected undera stereomicroscope and kept in MBS at 18 “C until use. Isolation of the germinal vesicles and preparation of oocyte fractions France. Tel.: 33-57-57-10-14; Fax: 33-57-57-10-15. 11 Present address: Dept. of Molecular Genetics, Institute of Tumor- were performed according to the procedures used by Solan and biology, Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Wien, Austria. ’ The abbreviation used is: MBS, modified Barth’s solution.

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RNase H Activities of X . laevis Oocytes

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Deutscher (33), except that thehomogenization of intact or enucleated oocytes was performed simply by repeated pipettingof 10 oocytes in 200 hybrid pl of J buffer (70 mM NH,Cl, 7 mM MgCl,, 0.1 mM EDTA, 2.5 mM --edenatured hybrid dithiothreitol, 10 mM Hepes, pH 7.4) through a yellow tip (0-200 pl) for pipettman (Gilson). Oocytes were induced to mature by addition of 1 PM progesterone to the external medium. Maturedoocytes were identified by the appearance of a typical white spotat the animalpole a few hours after addition of the hormone (34). Animal and vegetal halvesof matured and non-matured oocytes were obtained by using a procedure similar to the one described by Drummond et al. (351, and were homogenized a s described above with 10 pl of J buffer per oocyte half. Mitochondria and mitoplasts were isolatedfrom Xenopus oocytes as described by Brun et al. (36).Their purity was checked by measurement of the activity of marker enzymes, glucose-6-phosphate dehydrogenase (371, sulfite oxidase (38), and succinate cytochromes-reductase (39). Mitochondria were lysed in a buffer containing 0.5 M KC1 and 0.5% 0 5 10 15 20 Triton X-100, then dialyzed against 20 m~ potassium phosphate, pH 7.5, 2 mM 6-mercaptoethanol, and 20% glycerol (36). oocyte extract (PI) RNase HAssays-Labeled RNA.DNA hybrid substratesfor RNase H FIG.1. RNase H activity present in homogenates of stage VI were synthesized by in vitro transcription of heat-denatured calf thymus DNA or poly(dT) with E. coli RNA polymerase as described previ- oocytes. The hydrolytic activity was tested, on native (solid line) or RNA.DNA hybrid, according tothe liquid assay ously (40). Labeled RNA (.-lo00 nucleotides long) was synthesized by denatured (dotted line) phage SP6 polymerase transcription of a linearized plasmidDNA in the described under “Materials and Methods.” presence of [CX-~~PIATP. RNase H assays (liquid assays) were performedas described previ- respond to an activity similar t o the bovine RNase HIIa which ously (401, except that thebuffer used for testing activity contained 30 can degradesingle-stranded RNA as well as RNA.DNA hybrids mM Tris-HC1, pH 7.8, 10 mM (NH,),SO,, 2 mM MgCl,, and 0.01% p-mer(44).The Xenopus enzyme activity is divalent cation dependcaptoethanol. Under these conditions,1 unit of activity corresponds to ent. It can be optimally activated eitherby 2 mM magnesium or the amountof enzyme hydrolyzing 100 pmol of hybrid in 10 min at 37 “C 0.2 mM manganese (Fig. 2) indicating thepresence of a class I (23, 40). enzyme (Table I). The optimum of ammonium sulfate concenRenaturation gel assays (activity gels) were performed as described (40, 41) or with a procedure of increased sensitivity (42): it used a tration present in buffer was found to be 10 mM under magclassicalSDS-polyacrylamidegel,except that 32P-labeledRNA.DNA nesium but 20 mM under manganese. In these conditions, the substrate (.-300,000 cpm) was incorporated to the resolving gel before activities at optima are very similar (data not shown). In the polymerization. After electrophoresis,the gel wasprocessed to promote presence of magnesium the activity is inhibited by addition of protein renaturation and RNase H activity. Then the gel was fixed, manganese (Fig. 2, inset). Such behavior is either compatible extensively washed, and autoradiographed. Renatured proteins poswith thepresence of a class I1 enzyme, or corresponds to a class sessing RNase H activity were detected as white bands ona dark background. Ina control experiment, labeled RNA was incorporated in place I enzyme which would have a greater affinity for manganese than for magnesium but less activity under manganese than of the labeled hybrid to detect renaturable RNases activities. Neutralization assay was identical to the standard RNase H assay, under magnesium. Experiments described below support the except that samples were preincubated on ice for 45 min with the second interpretation. Eukaryotic RNasesH can be differentiantibody, before starting the reaction by addition of the RNA.DNA ated by their molecular weight, for instance by their sedimenhybrid (43). Antibodies were obtained and prepared as described by performed tation behavior (45)(Table I). Sedimentation studies Busen (43). Immunoprecipitations were done by mixing 80 pl of the with oocyte extracts revealed a single activity peak close t o the oocyte extract with 200 pl of protein A-Sepharose to which IgGs from bovine serum albumin marker andco-sedimenting with hemoimmune or control serum have been preadsorbed. After overnight incubation at 4 “C with gentle shaking the mixture was centrifuged in a globin (64kDa). No indication for a class I1 RNase H activity microcentrifuge for a few seconds and the RNase H activity of the was found (Fig. 3). supernatant determined. Immunoblots were performed as described Neutralizing antibodies raised against calf thymus RNase (40). HI do not neutralize thebovine class I1 activity. Those antibodUltracentrifugation Analysis-Oocyte extracts (200 pl) were centriies inhibit specifically the Xenopus enzyme present in oocytes, fuged at 250,000 x g for 66-68 h at 4 “C on 5-ml5-20% sucrose gradienucleated oocytes, and germinalvesicles as illustrated inFig. ents made ina 10 mM Tris-HC1, pH 7.8, buffer containing 0.5M KC1, 2 4. We also found in immunoprecipitation tests (see “Materials m~ EDTA, and 25% glycerol (23). The high salt concentration of the buffer used for centrifugation was intended to preventnonspecific pro- and Methods”) that the incubation of oocyte extracts with antein aggregation. Mitochondrial extracts were processed similarly, ex- tibodies from the immune serum preadsorbed t o protein Acept that the run was performed for only 15 h as the glycerol was Sepharosem completely depleted the extractfrom the RNase H omitted. Prior to load, samples were dialyzed against the buffer usedfor the run. Marker proteins (bovine serum albumin or hemoglobin) were activity, whereas no loss was observed with corresponding ansedimented in parallel runs. At the endof the run, fractions were col- tibodies of the control serum (data not shown). However, a lected from the bottom of the tube and the RNase H content was ana-second antiserum raised against the bovine class I enzymes lyzed using either the liquid assay or the renaturation gel assay as provided antibodies neutralizing the calf thymus class I enspecified. zyme activity (63)but not antibodies neutralizing theXenopus RESULTS

The Predominant RNase H of Xenopus Oocytes Is a Class I Enzyme-RNase H activity present in Xenopus oocytes can be readily determined by using a liquid assay (Fig. 1). No activity was seen on heat-denatured hybrids. This ruled out that the RNA strand could have been digested by a RNase specific for single-stranded RNA acting on the RNA strand of a n unwound hybrid. It shows also that the RNase H we are looking at is specific for RNA.DNA hybrids and consequently does not cor-

enzyme, suggesting that some antibodies of the first serum detect epitopesrelated to the active center of the bovine and the Xenopus enzyme, whereas antibodies of the second serum are directed to epitopes influencing only the active center of the bovine enzyme. In summary, all data argue for the predominant presence of a class I RNase H in oocyte extracts; they leave little space for the presence of a class I1 RNase H activity. In a further attempt todetect this activity, we came backto the procedure originally used to distinguish thetwo classes of enzymes and to separate them from each other, their different

RNase H Activities of X . laevis Oocytes

25187

100

“t- total

50

nucleus

Y -

O

5

15

10

[Me2+], mM

FIG.2. Dependence of the RNase H activity from X. laevis oocytehomogenatesondivalentcationsMeZ+. Assayswere performed with 1.5 1.11 of oocyte extract under conditions described under “Materials and Methods,” except that the concentration of divalent cations Mg2‘ (0)or Mn2+(0) was varied. Inset, inhibition of the X . laevis RNase Hactivity, tested under m 2M magnesium, by addition of increasing amounts of manganese chloride.

TABLEI Biochemical criteria used to distinguish the two classes of ribonucleases H DEAE binding CM binding Activation by Mg2 ions Activation ions by Mn2 Inhibition ions by Mn2 Molecular mass Sucrose density centrifugation Denaturing SDS-gel electrophoresis

Class I1

+

-

+ + -

200

100

kg IgG FIG.4. Neutralization of X.laevis RNase H activity presentin germinal vesicles (O), enucleated oocytes (O), or total oocytes (W) by a polyclonal antibody raised against calf thymus RNase HI. 100% corresponds to 0.5 enzyme units. c ,,

50

Class I

-

4 0

300

+ + -

+

67 kDa 45 kDa 68 kDa kDa 32

BSA

a

E 0 0

10

h

1 0

10

20

0

20

60

40

Fraction

80

number

FIG.5. Fractionation of X. laevis extract on DEAE cellulose. Extract (1.5 ml) of fully grown stage VI oocyte homogenate, obtainedas described under “Materials andMethods,” was dialyzed for 3 h against 1liter of buffer D(30 mM Tris-HCI, pH 7.8, containing 30%glycerol and 0.1% P-mercaptoethanol). 0.5 ml of the dialysate (4 mg/ml) was loaded on a 1.6 x 15-cm column filled with DE52 (Whatman) and equilibrated withbufferD (flow rate: 0.5 ml/min).After collection of the flowthrough, a linear 0-0.5 M KC1 gradient in buffer D was applied (A). The concentration of KC1 was determinedby conductimetry and fractions(1 ml) were tested for the presence of RNase H using the liquid assay described under “Materials and Methods”(0). 30

vianopoulos and Chargaff (47) (and discussed more recentlyby Busen and Frank (65) for RNases HI and HI‘ from calf thyFraction number mus), because the activities present in these 2 peaks have FIG.3. Sedimentation of the X.laevis RNase H activity on su- undistinguishabledivalentcationrequirements(datanot crose gradientas described under “Materials and Methods”. The arrow indicates the position of the bovine serum albumin (BSA) sedi- shown). Alternatively, one peak could correspond to a breakdown product of the nativeenzyme with different electrostatic mented in the same run under identical conditions. Fractions were numbered from the bottom to the top of the gradient and 10 p1 were properties, or both peaks to different states of post-translatested accordingly to the liquid assay as described under“Materia1s and tional modification of the sameenzyme. In a very last attempt Methods.” to detect classI1 RNase H activity,we looked for a possible loss of HI1 during the preparation of oocyte extracts. It has been binding toDEAE cellulose (22,46) (Table I). Whenthis experi- reported that the procedure of preparation of oocyte extracts ment was performed with Xenopus oocyte extract, all the ac- used inthis study can result in the loss of some basic proteins tivity was found to bind to the exchanger and none was recov- associated to yolk pellets during the centrifugation step (48). ered in the flow-through (Fig. 5). The presence of 2 peaks could We prepared extracts with the use of freon (trifluorochloroethrepresent 2 isoforms of the same enzyme as suggested by Stra- ane), a procedure reported tocircumvent the pitfallsmentioned

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RNase H Activities of X . laevis Oocytes

above (481,but we did not recover in these extracts activities significantly higher than those obtained by the classical procedure. Moreover the extraction of yolk pellets with freon did not permit more than 3% of the activity of the cell, whereas 10%of the oocyte non-yolk proteins were recovered in this step. The RNase H activity recovered from yolkpellets was not different from the bulk of the RNase H activity as judged by its sensitivity to divalent cations. In summary, there is no evidence for a specific loss of RNase HI1 during the preparation of the extracts. We conclude fromliquid assays that, apparently, oocytes contain only a class I RNase H activity, and at variance of all other eukaryotic cells do not seem to contain RNase HII. The RNase H Activity of the Oocyte Is Concentrated in the Germinal Vesicle-To analyze the relative RNase H activity levels of the cytoplasm and of the nucleus, Xenopus oocytes were enucleated as described under “Materials and Methods” and RNase H activity was determined and compared to that of total oocytes or isolated germinal vesicles. The enucleated oocytes serve as a source forpotential RNase H activities in the ooplasm. As shown in Fig. 6 A , the majority of oocyte enzyme activity is associated with the germinal vesicle. To ensure that the RNase H activity found in enucleated oocytes was not due to leakage of enzyme from the germinal vesicle at the time of enucleation, we compared the activity level of the animal half of an oocyte, whichcontains the germinal vesicle, with that of the vegetal half. The activity in extracts of the vegetal halves corresponded to the values found forenucleated oocytes (Fig. 6, B and A). This indicates that our enucleation procedure did not produce artifactual values, and that indeed a minor fraction of RNase H activity (-5%) is associated with the cytosolic fraction of Xenopus oocytes.As seen in Fig. 4, RNase H activity of total oocyte extracts, germinal vesicle extracts, and extracts from enucleated oocytes were neutralized by anti-bovine RNase HI antibodies in acomparable manner. Therefore, the activity present in the cytoplasm corresponds to RNase HI; around 5%of the oocyte RNase HI is localized in the cytoplasm and consequently 95% is present in the nucleus (experimental values found are lower, -65-70%, likely reflecting leakage of the enzyme during the procedure or perhaps also inaccuracies of the pipetteting in small volumes). This implies that the enzyme is more than 200-fold concentrated in thenucleus, as thisone has a volume of 40 nl and the diffusion-free compartment is about 0.5 pl for the stage VI oocyte (48).For comparison, Solan and Deutscher (33) have found in a very similar study that the tRNA-nucleotidyltransferaseis only 2 to 3 times concentrated in the nucleus relative to the cytosol.From simple calculations,’ the oocyte appears to contain the RNase H equivalent of 2.1-4.2 x lo5 somatic cells in accordance with previous estimates from J. B. Gurdon who has considered the oocyte to be roughly equivalent to 200,000 somatic cells (50) and from others who have found the oocyte to contain 100,000 times more DNA and RNA polymerases than a typical larval somatic cell (51). If we consider that thenuclear volume of calf thymocytes should be more or less similar to the one (113pm3)determined for human cells on the basis of an average nuclear diameter of about 6 pm (52), then thenuclear concentration of RNase HI in thymocytes is about 0.06 units/nl. The same calculation for the stage VI oocyte gives a nuclear concentration of 0.04-0.08 unitdnl, indicating that with the exception of its largesize, the oocyte is similar to somatic cells. Interestingly, after maturation and germinal vesicle breakdown, the RNase H level of the We have determined that one oocyte contains from 1.6 to 3.2 units of RNase HI depending of the batch of oocytes used (this reflects probably individual variations between frogs, and perhaps also seasonal variations). It has been previouslycalculated that 34 g of calf thymus (-136 billions of cells) contains about 1 million units of RNase HI (49).

8or

60

0

2

4

6

oocyte extract

8

10

8

10

(PI)

41

0

2

4

o o c y teex t r a c t

6

(PI)

FIG.6. Subcellular location of RNase H in Xenopus oocytes. Panel A, RNase H activities present in total oocytes (W), enucleated oocytes (O),and isolated germinal vesicles (0).“he cell equivalent was determined from the volume of extract used for the test (see “Materials and Methods”)as the numbers of oocytes, enucleated oocytes, or intact germinal vesicles used for preparing the extracts are known with precision as described under “Materials and Methods.” Panel B , RNase H activities present in totaloocytes (W), animal (O),and vegetal (0) halves ofXenopus oocytes.Panel C , RNase H activities present in totaloocytes (W), animal (O),and vegetal (0)halves of matured Xenopus oocytes.

vegetal half increases by a factor of 6 (around 30% of the total oocyte activity), indicating that a large part of the nuclear RNase HI isnot tightly associated with the chromatin fraction and therefore easily released (Fig. 6C). Attempts to Identify the Polypeptide(s)Supporting the RNase HIActiuity-We tried to detect those by immunoblotting using either the antiserum displaying IgGs neutralizing Xenopus RNase HI, or the non-neutralizing antiserum. We revealed four cross-reacting polypeptides with molecular masses of approximately 68,50,35, and 32 kDa with the neutralizing antiserum (Fig. 7). None was recognized by the control serum. Interest-

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nuclear factor to be detected in the liquid assay or, inversely, this 32-kDa protein is inhibited by a cytoplasmic factor. (ii)The 32-kDa polypeptide is a proteolytic product of the RNase HI 1 2 3 4 5 6 1 2 3 4 5 6 which is easily detected inan activity gel because short 116 polypeptides renature better than larger proteins as suggested 116 94 - 94 by Rong and Carl (41); the loss of a nuclear-targeting signal 68 - 68 after proteolysis could result in its accumulation in the cyto.. 60 - 60 plasm. (iii) The 32-kDa polypeptide corresponds to Xenopus 45 - 45 RNase HI1 which for unknown reasons remains undetected in 40 - 4 0 the liquid assay butis easily detected in thegel assay. (iv) The 32-kDa polypeptide corresponds to a new type of RNase H 25 - 25 activity not detected in the liquid assay. Additional observations described below give some support to the last two interpretations. We had considered enucleated oocytes to be equivalent tocytoplasm. This is certainly an oversimplification. Each oocyte contains about lo7 mitochondria FIG.7. Immunoblots of extracts of isolated germinal vesicles (53), and even if they are almost quantitatively pelleted with (lanes 1 and 2 ) , total oocytes (lanes 3 and 4 ) , and enucleated oocytes (lanes 5 and 6) probed with the non-neutralizing(panel yolk platelets in the 10,000 x g centrifugation step, we cannot A ) or neutralizingantibodies (panel B ) . Each lane was loaded with exclude that some have leaked during thehomogenization step 1 cell equivalent, except lunes 1 and 2 were loaded with 3 or 7 cell and that some RNase H found in the cytoplasm would be of equivalents, respectively. Germinal vesicles used for extract loaded in mitochondrial origin. With this in mind, we began to investilunes 1 and 2 correspond to the enucleated oocytes loaded in lunes 5 and 6,respectively. In lunes 3 and 4 extracts from 2 independent homogeni- gate theRNase H content of these organelles. They were purizations of total oocytes were loaded. Size markers are indicated to the fied from whole ovaries through differential centrifugation as left and right sides. described by Brun et al. (361, including the banding of mitochondria at the interface of 42.5 and 20% sucrose step gradiingly, the non-neutralizing antiserum recognized only three of ents. Mitoplasts were prepared by treating partof the purified those four polypeptides (peptides of 50, 35, and 32 kDa). mitochondria with digitonin to remove the mitochondrial exWhereas the proteins of 68,35, and32 kDa seem to be concen- ternal membrane. The purity of the mitochondria and the efitrated, if not exclusively found, in the germinal vesicle, the ciency of the digitonin treatment were checked by assaying BO-kDa protein seems to be preferentially located in the cyto- marker enzymes as described under “Materials and Methods.” plasm. We therefore consider that the 68-kDa protein is the Both the fresh lysate and the dialysate were tested for the best candidate for the Xenopus RNase HI: this protein was presence of RNase H activity by using the liquid assay. No recognized only by the neutralizing antiserum andis the most activity was found in these tests despite the use of large intensively decorated in germinal vesicles. In addition, this amounts of extract (upto 60 pg of mitochondrial proteins). This agrees with the molecular mass (64 kDa)found for the native implies that the activity found in enucleated oocytes is not of Xenopus RNase HI as determined by sedimentation analysis mitochondrial origin and is almost certainly cytosolic. However, (see above) and with the molecular mass estimated for the to our great surprise the 32-kDa activity band was very easily RNase HI from calf thymus (41). If these considerations are detected on activity gels loaded with mitochondrial extracts correct, then the RNase HI of Xenopus must be a monomeric (Fig. 10). This band corresponds to a mitochondrial protein and protein. So, one should expect to detect an activity in associa- does not originate from a contamination by cytosolic material tion with this protein band. However, we have never detected adhering to the external membrane of the mitochondria as this peptide in activity gels, even when using the more powerful equal amounts of mitochondria or mitoplasts yield a band of renaturation procedure that we have recently developed (42). similar intensity on these gels (Fig. 10). Our statement that But we have never loaded on those gels the very large amounts this 32-kDa protein is concentrated in mitochondria derives of enzyme that Rong and Carl (41) used for visualizing the from comparisons of the band intensities of known amounts of bovine class I monomer. Instead, in all activity gels, we have cytosolic and mitochondrial proteins loaded on the sameactivvery consistently detected a polypeptide of about 32 kDa and ity gel.3That thisRNase H activity remains undetected in the frequently also a shorter polypeptide of 28 kDa (Fig. 8A). When liquid assay could result from the presence of an inhibitor for using the more sensitive gel renaturation assaywe detected 2 this enzyme in mitochondria. This inhibitor should be macroadditional active polypeptides of 43 and 26 kDa (Fig. 8B, lane molecular as itis not removed bydialysis. So, we should rather 4 ). The easilydetected major polypeptide of 32 kDa was present suspect that the 32-kDa protein associates with another proin extractsof whole oocytesas well as inextracts of enucleated tein, possibly with itself, resulting ina lackof detectable RNase oocytes and isolated germinal vesicles. Surprisingly, the inten- H in theliquid assay. To test thishypothesis the sedimentation sity was not very different in lanes loaded with identical cell of this 32-kDa polypeptide in a sucrose gradient hasbeen folequivalents (1 oocyte) for all three extracts despite that the lowed. This band was found in fractions co-sedimenting with activity was at least 10 times higherin whole oocytescompared hemoglobin, whereas another part was found to sedimentmuch to enucleated oocytes. Even if the band intensity can be sus- faster (data not shown). This experiment shows that this propected not to be proportional to the activity loaded on the gel, it We have observed that the intensity produced by 10 pg of mitochonindicated that the amount of this polypeptide is not very much drial proteinsis equivalent tothe intensityproduced by the homogenate greater in the nucleus than in the cytosol. Indeed, a direct of one oocyte. As this amount of mitochondrial proteins has been esticomparison of the intensityof the 32-kDa band for individually mated to represent thetotal mitochondrial content of a stage VI oocyte enucleated oocytes to their germinal vesicle showed that this (54) it implies that there is as much of this 32-kDa protein in the polypeptide was found in higher amounts in the cytoplasm than cytoplasm as in mitochondria. It has been determined that an oocyte in thenucleus (Fig. 9). Thus, the pattern displayed by activity contains about lo7 mitochondria, each of an average volume of 0.3 pm3 (53) which gives a total volume of 3 nl to be compared to the estigels is apparently contradictory to the pattern deduced from mated 500 nl of the diffusion-free compartment of the whole oocyte(48). liquid assays. Such discrepancy could result from the following Therefore the 32-kDa protein is roughly concentrated 160 times in situations. (i)The 32-kDa polypeptide has to be associated to a mitochondria.

A

-

RNase H Activities of X. laevis Oocytes

25190

A 1

Im..

AL&a

FIG.8.Panel A, renaturation gel assay of 1 cell equivalent from total oocytes (lane 1 ), enucleated oocytes (lane 2 ) , and isolated germinal vesicles (lane 3).Renaturation in the gel was performed under 10 mM magnesium chloride (41).Panel B , renaturation gel assay of 1 cell equivalent of X . laeuis total oocyte extract (lanes 1 and 4 ) , 100 pg of proteins from purified mitochondria (lanes 2 and 5),and 2 units of E.coli RNase H (Promega)(lanes3 and 6 ) .Renaturation in the gel was performed under 10 mM magnesium chloride (lanes 1 3 )or 0.5 mM manganese chloride (lanes 4-6) (42).

2

B 1

3

2

3

4

5

6

kaia

43

-

30

-

30

-

20,l

-

14,4

94 67

20,l

94 67 43

14,4

001 GVEn

002 -

GV En

003 GV En

1

004 -

2

3

4

5

6

7

8

9

GVEn

32kDa

. " )

~

FIG.9.Renaturation gel assay analysis of oocytes individually (GV) and their dissected in their isolated germinal vesicles enucleated oocyte ( E n ) counterpart. Renaturation in the gel was performed under 10 m>l magnesium chloride (41).

tein is part of a dimer at least, andvery likely part of a larger multimeric complex. In an attemptto determine if this protein has a real preference for cleaving hybrids we performed an activity gel containing labeled RNA instead of labeled RNAeDNA hybrid. On this gel, in addition to marker proteins, we loaded 4 times side by side 10 pg of proteins of Xenopus crude extract and 10 pg of proteins of mitoplasts. After electrophoresis, the gel was cut in 4 parts and each part was renatured as usual but without divalent cation, or with 1mM CaCl,, 0.2 mM MnCl,, or 10 mM MgCl,. Surprisingly no RNase was detected in this assay, except a band of -39 kDa in thecrude extract for the partof the gel renatured with CaCl, (data not shown), and which may correspond to the recently described 36-kDa calcium-dependent RNase X (55).In conclusion, although not every substrate was tested, this mitochondrial 32-kDa corresponds likely to a genuine RNase H. DISCUSSION

The oocyte is a cell known to store many proteinsin view of their future utilization during embryonic development, and some nuclear proteins being stored in the cytoplasm of the oocyte before their migration into thenuclei of the developing

FIG.10. Renaturation gel assay comparing activities present in 1 cell equivalent of X . Laeuis oocyte (Lane 1) with 10 p g (lanes 2 and 3), 20 pg (lanes 4 and 5 ) ,30 pg (lanes 6 and 7),and 50 pg (lanes 8 and 9 )of proteins from mitochondria (Lanes2 , 4 , 6, and 8 ) or mitoplasts (lanes 3, 5, 7,and 9). Renaturation in the gel was performed under 0.5 mM manganese chloride (42).

embryo (56, 57). Thus, the RNase H of Xenopus oocytes could have been one of these proteins, but the resultsof the experiments reported in this paper clearly demonstrate that this is not the case. Comparisons with somatic cells indicate that the oocyte, and consequently the egg, has accumulated enough RNase HI for distributing it between its very rapidly dividing cells during embryogenesis without the need for new synthesis of this enzyme during cleavage, but thisstorage appears to be essentially nuclear. However, the 5% RNase HI activity remaining in the ooplasm are very likely responsible for the reported antisensemediated destruction of messenger RNAs. The presence of this enzyme in the cytoplasm can be the resultof several phenomenons. First, freshly synthesized enzymes on their way to the nucleus will contribute tothe cytoplasmic activity.As the translation rate in stage VI oocytes is very low (33, 50), it seems unlikely that this phenomenon would by itself account for the amount of activity found in the cytoplasm. Second, it could be the result of a progressive accumulation of proteolytic fragments of RNase HI, still enzymatically active but unable to return to the nucleus. Last butnot least, thisdistribution could simply reflect a steady-state equilibrium between proteins en-

RNase H Activities of X . laevis Oocytes tering the nucleus and leaving it. None of these contributions are a priori exclusive from each other, but their respective importance is hard to estimateat present. An additional natural process which would increase the amountof the enzyme in the cytoplasm, namely cell division, could not account for the presence of cytoplasmic RNase H in the Xenopus oocytes as these cells are blocked in meiotic prophase from the very beginning of oogenesis, a process which takes months toprovide fully grown stage VI oocytes. However, when meiosis is induced to resume by treatment of the oocytes with progesterone, then we found that the enzyme distributes itself all over the cell in a way similar to thatobserved in somatic cells. The conclusion is that in each of these cells the RNase HI does not seem tightly linked t o chromatin and remainsfree to diffuse. The apparentlack of class I1 RNase H activityin theoocyte, as inferred from assays in solution is surprising as it would implyxenopus tobe a n exception among thevery different and phylogenetically distant organisms such as Crithidia fasciculata (23) and Homo sapiens4 which all contain the class I1 RNase H. As the RNase HI1 is thought to be involved in transcription, and as the early embryo is transcriptionally silent, the lack of RNase HI1 in the last stage of oogenesis could have been related to this particular physiological state. However, studies on eggs and embryos did not show evidence for the appearance of RNase HI1 activity during development, in particular on embryos after the mid-blastula where transcription h a s r e ~ u m e dPreliminary .~ fractionation of Xenopus liver extract on DEAE columns has also failed to find any class I1 RNase H activity (data not shown). Contrasting with the conclusion of liquid assays, the picture obtained using the renaturation gel assay is much more complex. At least four polypeptides are detected in thisassay. The predominant one has an apparent molecular mass of 32 kDa which is exactly the one reported for a proteolytic product of mammalian RNase HI (41),for a protein with the characteristics of RNase HI1 in Krebs ascites cells (58), and for well characterized mammalian RNasesHII.4 The finding thatthis polypeptide is highly concentrated inmitochondria and almost absent from the ooplasm and that itspresence remains undetected by the classical liquid assay incited us to hypothesize that this polypeptide may correspond to the “missing” RNase HII. Why the activity of this peptide is not detected in theliquid assay is not precisely known. Our sedimentation studies suggest that it could be the result from the participation of this “cryptic RNaseHII” to a larger multimericcomplex, perhaps as a part of a n RNA polymerase as shown for the 49-kDa subunit of yeast RNA polymerase (59). This property could also explain why the 32 kDa was notdetected in theflow-through fractions of the DEAE column (data not shown). If this interpretationis correct, this would suggest that detectable RNase HI1 activity in tissuesfrom other organisms may infact be the result from a higher propensity of this polypeptide to dissociate from its physiological multimeric complex. Using liquid assays, Soriano et al. (60) detected the presence of two RNase H activities, with properties similar to RNases HI and 11, in mitochondria from brains of chick embryos, but as their has been no tests on mitoplasts, one cannot exclude that these activities resulted from cytosolic contaminations. More recently, several observations concerning mitochondrial proteins possessing RNase H activities have been reported. In Neurospora, an RNase H activity is present inmitochondrial nucleoprotein complexes, but its molecular mass is only 25 kDa, as determined on activity P. Frank, S. Albert, C. Cazenave, J. J. and ToulmB, unpublished data. Frank, P. (1986) EuKaryontenRibonucleasen HI. DiplomArbeit, Fakultat fur Chemie und Pharmazie, Universitat Tubingen, Germany.

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gels, and itsactivity is readily detectablein solution witheither Mg2‘ or Mn2+(61). In vertebrates, a low abundance endonuclease, Endo G, has been found to possess RNase H activity in addition t o DNase and RNase activities (62). It is distributed in both the nucleus and themitochondria, acts asa homodimer of -26-28 kDa but is synthesized as a precursor of -32 kDa. Even if this size corresponds to the one observed in our gels it remains hard to understand why the precursor would be so abundant inside themitochondria. None of these proteins has properties described for a eukaryotic class I1 RNase H. Future work on purifiedmitochondria from varioussourcesshould help to obtaina better understandingof the statusof RNase H activities in these organelles. Acknowledgment-We thank EMBO for a short-term fellowship which allowed C. C. to start this work during his stay in Tubingen. REFERENCES 1. Stein, H., and Hausen, P. (1969) Science 166, 393-395 2. Hausen, P., and Stein, H. (1970) Eur. J. Biochem. 14, 27%283 3. Moelling, K., Bolognesi, D. P., Bauer, H., Biisen, W., Plassmann, H. W., and Hausen, P. (1971) Nut. New Biol. 234, 240-243 4. Henry, C.M., Ferdinand, F. J., and Knippers, R.(1973) Biochem. Biophys. Res. Commun. 50,603411 5. Miller, H. I.,Riggs, A. D., and Gill, G.N. (1973) J. Biol. Chem. 248,2621-2624 6. Berkower, I., Leis, J., and Hunvitz, J. (1973) J. Biol. Chem. 248, 5914-5921 7. Wyers, F.,Sentenac,A.,and Fromageot, P. (1973)Eur. J. Biochem. 35,270-281 8. Sawai, Y., Unno, M., and Tsukada,K. (1978) Biochem. Biophys. Res. Commun. 84,313-321 9. Sawai, Y., Sawasaki, Y., and Tsukada, K. (1977) Life Sci. 21, 1351-1356 10. OCuinn, G., Persico, F.J., and Gottlieb, A. A. (1973) Biochim. Biophys. Acta 324,78-85 11. Sarngadharan, M. G., Leis, J. P., and Gallo, R. C. (1975) J. Biol. Chem. 250, 365373 12. Sawai, Y., Sugano, N., and Tsukada, K. (1978) Biochim. Biophys. Acta 518, 181-185 13. Crouch, R.J., and Dirksen, M. L. (1982) in Nucleases (Linn, S. M., and Robert, R. J., eds) pp. 211-241, Cold Spring HarborLaboratory,Cold Spring Harbor, NY 14. Crouch, R. J. (1990) The New Biologist 2, 771-777 15. Kohlstaedt, L. A., Wang, J., Friedmann, J. M., Rice, P. A., and Steitz, T.A. (1992) Science 256, 1783-1790 16. Fu, T. B., and Taylor, J. (1992) J. Virol. 66, 4271-4278 17 Ben-Artzi, H. B.,~Zeelon,E., h i t , B., Wortzel, A., Gorecki, M., and Panet, A. (1993) J. Biol. Chem. 268, 16465-16471 18 Ogawa, T., Pickett, G. G., Kogoma, T., andKornberg,A. (1984) Proc. Natl. Acad. Sci. U. S. A . 81, 1040-1044 19 Dasgupta, S., Masukata, H., and ’Ibmizawa,J. (1987) Cell 51, 1113-1122 20 Katayanagi, IC, Miyagawa, M., Matsushima, M., Ishikawa, S., Kanaya, S., Ikehara, M., Matsuzaki, T., and Morikawa, K. (1990) Nature 347,306-309 21 Yang, W., Hendrickson, W. A,, Crouch, R.J., and Satow, Y. (1990) Science 249, 139%1405 22 Vonwirth, H., Kock, J., and Biisen, W.(1991) Experientia (Basel)47,92-95 23 Biisen, W., Peters, J. H., and Hausen, P. (1977) Eur. J. Biochem. 74,203-208 24 Cazenave, C., and H&ne C. (1991) in Antisense Nucleic Acids and Proteins; (Mol,J. N. M., and Van der Krol,A. R., eds) Fundamentals and Applications Marcel Dekker Inc., New York 25 Cazenave, C., Loreau, N., Thuong, N. T., ToulmB, J. J., and HBlBne, C. (1987) Nucleic Acids Res. 15, 47174736 26 Dash, P., Lotan, I., Knapp, M., Kandel, E. R., and Goelet, P. (1987) Proc. Natl. Acad. Sci. U. S. A . 84, 7896-7900 27 Bertrand, J. R., Imbach, J. L., Paoletti, C., andMalvy,C. (1989) Biochem. Biophys. Res. Commun. 164, 311-318 28. Boiziau, C., Kurfurst, R., Cazenave, C., Roig, V.,Thuong, N. T., and ToulmB,J. J. (1991) Nucleic Acids Res. 19, 1113-1119 29. Eder, P. S. and Walder, J. A. (1991) J. Biol. Chem. 266, 64724479 30. Eder, P. S., Walder, R. Y., and Walder, J. A. (1993) Biochimie (Paris) 75, 123-126 31. Colman, A. (1984) in Dunscription and Dunslation-A Practical Approach (Hames, E. D., ed) pp. 271-302, IRL Press, Oxford 32. Dumont, J.N. (1972) J. Morphol. 136, 153-180 33. Solan, A., and Deutscher, M. P. (1982) Nucleic Acids Res. 14, 4397-4407 34. Sagata, N., Oskarsson,M., Copeland,T., Brumbaugh, G., and Vande Woude,G . F.(1988) Nature 335,519-525 35. Drummond, D. R.,Annstrong,J.,and Colman,A. (1985)Nucleic AcidsRes.13, 7375-7394 36. Brun, G.,Vannier, P.,Scovassi, I., and Callen, J. C. (1981)Eur:J. Biochm. 118, 407415 37. Langdon, R.G. (1966) Methods Enzymol. 9,126-131 38. Cohen, H. J., Betcher-Lange,S., Kessler, D. L., and Rajagopalan, K., V (1972) J. Biol. Chem. 247, 7759-7766 39. Sottocasa, G. L., Kuylenstierna, B., Ernster, L., and Bergstrand,A. (1967) J . Cell Biol. 32, 415-438 40. Casenave, C., Frank, E, and Biisen, W. (1993) Biochimie (Paris) 75, 113-122 41. Rong, Y.W., and Carl, P.L. (1990) Biochemistry 29, 383-389

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