Characterization and subcellular localization of ribonuclease H

0 1994 by The American Society for lb JOURNAL OF BIOL~CICAL C m a n m ~

Biochemistry and Molecular Bioloa, Inc Vol. 269, No. 40, Issue of October 7, pp. 25185-25192, 1994

Printed in U.S.A.

Characterization and Subcellular Localization of Ribonuclease H Activities from Xenopus Zaevis Oocytes*

(Received for publication, 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- nopus oocytes were investigated by using either liquid assays or renaturation gel assays. Whereas the test in solution detected an apparently unique class I ribonuclease H activity, the activity gels did not detect this enzyme but another one with the molecular weight expected for a class I1 ribonuclease H. The ribonuclease HI was found to be primarily concentrated in the germinal vesicle, but around 5% of this activity was detected in the cytoplasm and may correspond to the activity involved in antisense oligonucleotide-mediated destruction of messenger RNAs. The concentration of this class I ribonuclease H in oocytes is similar to that in somatic cells. The class I1 ribonuclease H remained undetectable by the test in solution because its activity was cryptic. On activity gel, a polypeptide with the apparent molecular mass of 32 ma , expected for a ribonuclease HII, was found to be concentrated in mitochondria although no RNase H activity could be detected by using the liquid assay. Based on sedimentation studies, we hypothesize that the apparent absence of RNase H activity in solution could be the result of the association of this 32-kDa polypeptide with other polypeptides, or possibly nucleic acids, to form a multimer of, until now, unknown function.

Ribonucleases H (RNases H for short) are RNA-degrading enzymes active only on RNA hybridized to a complementary DNA strand. This activity has been first identified in calf thymus (1, 2) and soon afterwards in a retrovirus in association with reverse transcriptase (3). Thereafter similar activities were recognized in Escherichia coli cells (4-6) and in a great number of eukaryotic cells of various origins, as for example, yeast (71, rat liver (81, and rat brain (91, tumor cells (10,111, and plant cells (12) (for reviews, see Refs. 13 and 14 and Crouch and Toulme (64)).

Despite the fact that these enzymes have been first found more than 20 years ago in cells of higher eukaryotes their exact function in nucleic acid metabolism is still largely unknown. In contrast, a much better knowledge of the structure and fmc- tion of the RNase H associated with reverse transcriptase has accumulated in the last years (15) and the role of RNase H activity in the whole process of reverse transcription is now

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked

indicate this fact. “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to

$ Charge de Recherches at CNRS. To whom correspondence should be addressed: Laboratoire de Biophysique MolBculaire, INSERM U386, Universite de Bordeaux 11,146 rue Leo Saignat, 33076 Bordeaux Cedex, France. Tel.: 33-57-57-10-14; Fax: 33-57-57-10-15.

11 Present address: Dept. of Molecular Genetics, Institute of Tumor- biology, Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Wien, Austria.

known in great detail (16, 17). Similarly, numerous genetic studies performed mostly with E. coli have demonstrated the role of RNase HI in the precise initiation of DNA replication of the bacterial chromosome (18) or plasmids (19); moreover the crystal structure of this enzyme has been recently determined (20, 21). Although the detailed structure and function of the eukaryotic enzymes is unknown, a general picture can be drawn from the various attempts to characterize these enzymes further: all cells from lower and higher eukaryotes so far tested are known to contain at least two forms of RNase H activity, a class I and a class I1 ribonuclease H (22). These enzymes can be differentiated by physical, biochemical, and serological parameters and they are believed to function during DNA replication and RNA transcription, respectively (23). These enzymes play a critical role in the success of the so-called “hybrid arrest of translation” experiments using antisense oli- godeoxynucleotides (24). This has been shown in cell-free extracts. Xenopus oocytes are the only cells in which the involve- ment of RNase H in antisense effect has been clearly established (25, 26). Indeed, chemically modified oligos which do not allow RNase H activity fail to inhibit translation unless the cap site or the initiation codon region are targeted (27,281. Interestingly, the presence of RNase H activity in the cytoplasm ofXenopus oocytes has been indirectly proved: antisense oligonucleotides induced the selective degradation of mature mRNAs injected in the cytoplasm (25). The cytoplasmic localization of this activity had to be explored further as this finding was rather unexpected in view of the putative functions of eukaryotic RNases H in replication (231, transcription (231, or DNA repair (29, 30) which would have rather argued for a nuclear localization. As this cell is arrested in meiotic prophase and is known to store in the cytoplasm some proteins which will migrate to the nuclei later during embryonic development it was of interest to investigate whether RNase H belongs to this set of maternal proteins.

In an attempt to obtain a better knowledge about the participation of RNases H in antisense-mediated arrest of translation, we started investigating the nature and subcellular localization of the RNase H activities present in Xenopus oocytes.

MATERIALS AND METHODS Xenopus Oocytes-Xenopus laeuis frogs (purchased from Centre de

Recherches de Biochimie Macromoleculaire, CNRS in Montpellier) were anesthesized with 1 g/liter MS222 (Sandoz) and their ovaries surgically removed, dilacerated in modified Barth’s saline (MBS)’ (31), and the oocytes defolliculated at room temperature under constant gentle stir- ring by treatment with 0.4 mglml dispase in MBS for 4 h, then 1 mg/ml collagenase in MBS for 1 h. Thereafter the oocytes were extensively washed with MBS. Fully grown stage VI oocytes (32) were manually selected under a stereomicroscope and kept in MBS at 18 “C until use.

Isolation of the germinal vesicles and preparation of oocyte fractions were performed according to the procedures used by Solan and

’ The abbreviation used is: MBS, modified Barth’s solution.

25185

25186 RNase H Activities of X. laevis Oocytes Deutscher (33), except that the homogenization of intact or enucleated oocytes was performed simply by repeated pipetting of 10 oocytes in 200 pl of J buffer (70 mM NH,Cl, 7 mM MgCl,, 0.1 mM EDTA, 2.5 mM 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. Matured oocytes were identified by the appearance of a typical white spot at the animal pole a few hours after addition of the hormone (34).

Animal and vegetal halves of matured and non-matured oocytes were obtained by using a procedure similar to the one described by Drum- mond et al. (351, and were homogenized as described above with 10 pl of J buffer per oocyte half.

Mitochondria and mitoplasts were isolated from 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% Triton X-100, then dialyzed against 20 m~ potassium phosphate, pH 7.5, 2 mM 6-mercaptoethanol, and 20% glycerol (36).

RNase HAssays-Labeled RNA.DNA hybrid substrates for RNase H were synthesized by in vitro transcription of heat-denatured calf thymus DNA or poly(dT) with E. coli RNA polymerase as described previously (40). Labeled RNA (.-lo00 nucleotides long) was synthesized by phage SP6 polymerase transcription of a linearized plasmid DNA in the presence of [CX-~~PIATP.

RNase H assays (liquid assays) were performed as described previously (401, except that the buffer used for testing activity contained 30 mM Tris-HC1, pH 7.8, 10 mM (NH,),SO,, 2 mM MgCl,, and 0.01% p-mercaptoethanol. Under these conditions, 1 unit of activity corresponds to the amount of enzyme hydrolyzing 100 pmol of hybrid in 10 min at 37 “C (23, 40).

Renaturation gel assays (activity gels) were performed as described (40, 41) or with a procedure of increased sensitivity (42): it used a classical SDS-polyacrylamide gel, except that 32P-labeled RNA.DNA substrate (.-300,000 cpm) was incorporated to the resolving gel before polymerization. After electrophoresis, the gel was processed to promote protein renaturation and RNase H activity. Then the gel was fixed, extensively washed, and autoradiographed. Renatured proteins possessing RNase H activity were detected as white bands on a dark back- ground. In a control experiment, labeled RNA was incorporated in place of the labeled hybrid to detect renaturable RNases activities.

Neutralization assay was identical to the standard RNase H assay, except that samples were preincubated on ice for 45 min with the antibody, before starting the reaction by addition of the RNA.DNA hybrid (43). Antibodies were obtained and prepared as described by Busen (43). Immunoprecipitations were done by mixing 80 pl of the oocyte extract with 200 pl of protein A-Sepharose to which IgGs from immune or control serum have been preadsorbed. After overnight incubation at 4 “C with gentle shaking the mixture was centrifuged in a microcentrifuge for a few seconds and the RNase H activity of the

(40). supernatant determined. Immunoblots were performed as described

Ultracentrifugation Analysis-Oocyte extracts (200 pl) were centrifuged at 250,000 x g for 66-68 h at 4 “C on 5-ml5-20% sucrose gradi- ents made in a 10 mM Tris-HC1, pH 7.8, buffer containing 0.5 M KC1, 2 m~ EDTA, and 25% glycerol (23). The high salt concentration of the buffer used for centrifugation was intended to prevent nonspecific protein aggregation. Mitochondrial extracts were processed similarly, except that the run was performed for only 15 h as the glycerol was omitted. Prior to load, samples were dialyzed against the buffer used for the run. Marker proteins (bovine serum albumin or hemoglobin) were sedimented in parallel runs. At the end of the run, fractions were col- lected from the bottom of the tube and the RNase H content was ana- lyzed using either the liquid assay or the renaturation gel assay as specified.

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 an unwound hybrid. It shows also that the RNase H we are looking at is specific for RNA.DNA hybrids and consequently does not cor-

hybrid

--e- denatured hybrid

0 5 10 15 20

oocyte extract (PI)

FIG. 1. RNase H activity present in homogenates of stage VI oocytes. The hydrolytic activity was tested, on native (solid line) or denatured (dotted line) RNA.DNA hybrid, according to the liquid assay described under “Materials and Methods.”

respond to an activity similar to the bovine RNase HIIa which can degrade single-stranded RNA as well as RNA.DNA hybrids (44). The Xenopus enzyme activity is divalent cation depend- ent. It can be optimally activated either by 2 mM magnesium or 0.2 mM manganese (Fig. 2) indicating the presence of a class I enzyme (Table I). The optimum of ammonium sulfate concentration present in buffer was found to be 10 mM under magnesium but 20 mM under manganese. In these conditions, the activities at optima are very similar (data not shown). In the presence of magnesium the activity is inhibited by addition of manganese (Fig. 2, inset). Such behavior is either compatible with the presence of a class I1 enzyme, or corresponds to a class I enzyme which would have a greater affinity for manganese than for magnesium but less activity under manganese than under magnesium. Experiments described below support the second interpretation. Eukaryotic RNases H can be differentiated by their molecular weight, for instance by their sedimentation behavior (45) (Table I). Sedimentation studies performed with oocyte extracts revealed a single activity peak close to the bovine serum albumin marker and co-sedimenting with hemoglobin (64 kDa). No indication for a class I1 RNase H activity was found (Fig. 3).

Neutralizing antibodies raised against calf thymus RNase HI do not neutralize the bovine class I1 activity. Those antibodies inhibit specifically the Xenopus enzyme present in oocytes, enucleated oocytes, and germinal vesicles as illustrated in Fig. 4. We also found in immunoprecipitation tests (see “Materials and Methods”) that the incubation of oocyte extracts with antibodies from the immune serum preadsorbed to protein A- Sepharosem completely depleted the extract from the RNase H activity, whereas no loss was observed with corresponding antibodies of the control serum (data not shown). However, a second antiserum raised against the bovine class I enzymes provided antibodies neutralizing the calf thymus class I enzyme activity (63) but not antibodies neutralizing the Xenopus enzyme, suggesting that some antibodies of the first serum detect epitopes related 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 to detect this activity, we came back to the procedure originally used to distinguish the two classes of enzymes and to separate them from each other, their different

RNase H Activities of X. laevis Oocytes 25187

Y -

O 5 10 15

[Me2+], mM

FIG. 2. Dependence of the RNase H activity from X. laevis oo-

formed with 1.5 1.11 of oocyte extract under conditions described under cyte homogenates on divalent cations MeZ+. Assays were per-

“Materials and Methods,” except that the concentration of divalent cations Mg2‘ (0) or Mn2+ (0) was varied. Inset, inhibition of the X . laevis RNase H activity, tested under 2 mM magnesium, by addition of increas- ing amounts of manganese chloride.

TABLE I Biochemical criteria used to distinguish the two

classes of ribonucleases H Class I Class I1

DEAE binding + - CM binding - + Activation by Mg2 ions + + Activation by Mn2 ions + - Inhibition by Mn2 ions - + Molecular mass Sucrose density centrifugation 67 kDa 45 kDa Denaturing SDS-gel electrophoresis 68 kDa 32 kDa

a 0 0 - E 10 1

BSA

h 0 10 20 30

Fraction number

FIG. 3. Sedimentation of the X. laevis RNase H activity on sucrose gradient as described under “Materials and Methods”. The arrow indicates the position of the bovine serum albumin (BSA) sedimented in the same run under identical conditions. Fractions were numbered from the bottom to the top of the gradient and 10 p1 were tested accordingly to the liquid assay as described under“Materia1s and Methods.”

binding to DEAE cellulose (22,46) (Table I). When this experiment was performed with Xenopus oocyte extract, all the activity was found to bind to the exchanger and none was recovered in the flow-through (Fig. 5). The presence of 2 peaks could represent 2 isoforms of the same enzyme as suggested by Stra-

100

“t- total 50 nucleus

4 0 100 200

kg IgG FIG. 4. Neutralization of X. laevis RNase H activity present in

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.

50 c 300 , ,

0 20 40 60 80

Fraction number

FIG. 5. Fractionation of X. laevis extract on DEAE cellulose. Extract (1.5 ml) of fully grown stage VI oocyte homogenate, obtained as described under “Materials and Methods,” was dialyzed for 3 h against 1 liter 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 with buffer D (flow rate: 0.5 ml/min). After collection of the flow- through, a linear 0-0.5 M KC1 gradient in buffer D was applied (A). The concentration of KC1 was determined by conductimetry and fractions (1

described under “Materials and Methods” (0). ml) were tested for the presence of RNase H using the liquid assay

vianopoulos and Chargaff (47) (and discussed more recently by Busen and Frank (65) for RNases HI and HI‘ from calf thymus), because the activities present in these 2 peaks have undistinguishable divalent cation requirements (data not shown). Alternatively, one peak could correspond to a breakdown product of the native enzyme with different electrostatic properties, or both peaks to different states of post-transla- tional modification of the same enzyme. In a very last attempt to detect class I1 RNase H activity, we looked for a possible loss of HI1 during the preparation of oocyte extracts. It has been reported that the procedure of preparation of oocyte extracts used in this study can result in the loss of some basic proteins associated to yolk pellets during the centrifugation step (48). We prepared extracts with the use of freon (trifluorochloroeth- ane), a procedure reported to circumvent the pitfalls mentioned

25188 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 yolk pellets 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 from liquid 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 for potential 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, which contains the germinal vesicle, with that of the vegetal half. The activity in extracts of the vegetal halves cor- responded to the values found for enucleated 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 a comparable 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 the nucleus, as this one 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-nucleotidyltransferase is only 2 to 3 times concentrated in the nucleus relative to the cytosol. From simple calcula- tions,’ the oocyte appears to contain the RNase H equivalent of 2.1-4.2 x lo5 somatic cells in accordance with previous esti- mates from J. B. Gurdon who has considered the oocyte to be roughly equivalent to 200,000 somatic cells (50) and from oth- ers 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 the nuclear volume of calf thymocytes should be more or less similar to the one (113 pm3) determined for human cells on the basis of an average nuclear diameter of about 6 pm (52), then the nuclear 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 large size, the oocyte is similar to somatic cells. Interestingly, after matura- tion 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 previously calculated that 34 g of calf thymus (-136 billions of cells) contains about 1 million units of RNase HI (49).

60 8or 0 2 4 6 8 10

oocyte extract (PI)

41

0 2 4 6 8 10

oocyte extract (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

germinal vesicles used for preparing the extracts are known with pre- and Methods”) as the numbers of oocytes, enucleated oocytes, or intact

cision as described under “Materials and Methods.” Panel B , RNase H activities present in total oocytes (W), animal (O), and vegetal (0) halves ofXenopus oocytes. Panel C, RNase H activities present in total oocytes (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 is not 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 approxi- mately 68,50,35, and 32 kDa with the neutralizing antiserum (Fig. 7). None was recognized by the control serum. Interest-


A 1 2 3 4 5 6

116 - 94 - 68 - 60 - 45 - 40 -

25 -

1 2 3 4 5 6

- 116 - 94

68 60

- 45 40

. . - - -

-

- 25

FIG. 7. Immunoblots of extracts of isolated germinal vesicles (lanes 1 and 2) , total oocytes (lanes 3 and 4 ) , and enucleated oocytes (lanes 5 and 6) probed with the non-neutralizing (panel A) or neutralizing antibodies (panel B ) . Each lane was loaded with 1 cell equivalent, except lunes 1 and 2 were loaded with 3 or 7 cell equivalents, respectively. Germinal vesicles used for extract loaded in lunes 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- zations of total oocytes were loaded. Size markers are indicated to the left and right sides.

ingly, the non-neutralizing antiserum recognized only three of those four polypeptides (peptides of 50, 35, and 32 kDa). Whereas the proteins of 68,35, and 32 kDa seem to be concentrated, if not exclusively found, in the germinal vesicle, the BO-kDa protein seems to be preferentially located in the cytoplasm. We therefore consider that the 68-kDa protein is the best candidate for the Xenopus RNase HI: this protein was recognized only by the neutralizing antiserum and is the most intensively decorated in germinal vesicles. In addition, this agrees with the molecular mass (64 kDa) found for the native Xenopus RNase HI as determined by sedimentation analysis (see above) and with the molecular mass estimated for the RNase HI from calf thymus (41). If these considerations are correct, then the RNase HI of Xenopus must be a monomeric protein. So, one should expect to detect an activity in association with this protein band. However, we have never detected this peptide in activity gels, even when using the more powerful renaturation procedure that we have recently developed (42). But we have never loaded on those gels the very large amounts of enzyme that Rong and Carl (41) used for visualizing the bovine class I monomer. Instead, in all activity gels, we have very consistently detected a polypeptide of about 32 kDa and frequently also a shorter polypeptide of 28 kDa (Fig. 8A). When using the more sensitive gel renaturation assay we detected 2 additional active polypeptides of 43 and 26 kDa (Fig. 8B, lane 4 ). The easily detected major polypeptide of 32 kDa was present in extracts of whole oocytes as well as in extracts of enucleated oocytes and isolated germinal vesicles. Surprisingly, the intensity was not very different in lanes loaded with identical cell equivalents (1 oocyte) for all three extracts despite that the activity was at least 10 times higher in whole oocytes compared to enucleated oocytes. Even if the band intensity can be sus- pected not to be proportional to the activity loaded on the gel, it indicated that the amount of this polypeptide is not very much greater in the nucleus than in the cytosol. Indeed, a direct comparison of the intensity of the 32-kDa band for individually enucleated oocytes to their germinal vesicle showed that this polypeptide was found in higher amounts in the cytoplasm than in the nucleus (Fig. 9). Thus, the pattern displayed by activity gels is apparently contradictory to the pattern deduced from liquid assays. Such discrepancy could result from the following situations. (i) The 32-kDa polypeptide has to be associated to a

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 which is easily detected in an activity gel because short polypeptides renature better than larger proteins as suggested by Rong and Carl (41); the loss of a nuclear-targeting signal after proteolysis could result in its accumulation in the cytoplasm. (iii) The 32-kDa polypeptide corresponds to Xenopus RNase HI1 which for unknown reasons remains undetected in the liquid assay but is easily detected in the gel assay. (iv) The 32-kDa polypeptide corresponds to a new type of RNase H 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 to cytoplasm. This is certainly an over- simplification. Each oocyte contains about lo7 mitochondria (53), and even if they are almost quantitatively pelleted with yolk platelets in the 10,000 x g centrifugation step, we cannot exclude that some have leaked during the homogenization step and that some RNase H found in the cytoplasm would be of mitochondrial origin. With this in mind, we began to investigate the RNase H content of these organelles. They were purified from whole ovaries through differential centrifugation as described by Brun et al. (361, including the banding of mitochondria at the interface of 42.5 and 20% sucrose step gradi- ents. Mitoplasts were prepared by treating part of the purified mitochondria with digitonin to remove the mitochondrial external membrane. The purity of the mitochondria and the efi- ciency of the digitonin treatment were checked by assaying marker enzymes as described under “Materials and Methods.” Both the fresh lysate and the dialysate were tested for the presence of RNase H activity by using the liquid assay. No activity was found in these tests despite the use of large amounts of extract (up to 60 pg of mitochondrial proteins). This implies that the activity found in enucleated oocytes is not of mitochondrial origin and is almost certainly cytosolic. However, to our great surprise the 32-kDa activity band was very easily detected on activity gels loaded with mitochondrial extracts (Fig. 10). This band corresponds to a mitochondrial protein and does not originate from a contamination by cytosolic material adhering to the external membrane of the mitochondria as equal amounts of mitochondria or mitoplasts yield a band of similar intensity on these gels (Fig. 10). Our statement that this 32-kDa protein is concentrated in mitochondria derives from comparisons of the band intensities of known amounts of cytosolic and mitochondrial proteins loaded on the same activity gel.3 That this RNase H activity remains undetected in the liquid assay could result from the presence of an inhibitor for this enzyme in mitochondria. This inhibitor should be macro- molecular as it is not removed by dialysis. So, we should rather suspect that the 32-kDa protein associates with another protein, possibly with itself, resulting in a lack of detectable RNase H in the liquid assay. To test this hypothesis the sedimentation of this 32-kDa polypeptide in a sucrose gradient has been fol- lowed. This band was found in fractions co-sedimenting with hemoglobin, whereas another part was found to sediment much faster (data not shown). This experiment shows that this pro-

We have observed that the intensity produced by 10 pg of mitochondrial proteins is equivalent to the intensity produced by the homogenate of one oocyte. As this amount of mitochondrial proteins has been estimated to represent the total mitochondrial content of a stage VI oocyte (54) it implies that there is as much of this 32-kDa protein in the cytoplasm as in mitochondria. It has been determined that an oocyte 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 esti-

Therefore the 32-kDa protein is roughly concentrated 160 times in mated 500 nl of the diffusion-free compartment of the whole oocyte (48).

mitochondria.

25190 RNase H Activities of X. laevis Oocytes

of 1 cell equivalent from total oocytes FIG. 8. Panel A, renaturation gel assay

(lane 1 ), enucleated oocytes (lane 2), and isolated germinal vesicles (lane 3). Rena- turation 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) (lanes 3 and 6) . Renaturation in the gel was performed under 10 mM magnesium chloride (lanes

4-6) (42). 1 3 ) or 0.5 mM manganese chloride (lanes

001 002

En GV En GV - -

A

Im.. 1 2 3 AL&a

94

67

43

30

20,l

14,4

003 004

En GV En GV - -

FIG. 9. Renaturation gel assay analysis of oocytes individually dissected in their isolated germinal vesicles (GV) and their 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, and very likely part of a larger multimeric complex.

In an attempt to determine if this protein has a real prefer- ence 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 1 mM CaCl,, 0.2 mM MnCl,, or 10 mM MgCl,. Surprisingly no RNase was detected in this assay, except a band of -39 kDa in the crude extract for the part of the gel renatured with CaCl, (data not shown), and which may correspond to the recently described 36-kDa calcium-depend- ent 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 proteins in view of

their future utilization during embryonic development, and some nuclear proteins being stored in the cytoplasm of the oocyte before their migration into the nuclei of the developing

32kDa .")

B

1 2 3 4 5 6 kaia

- 94 - 67 - 43

- 30

- 20,l

- 14,4

1 2 3 4 5 6 7 8 9

FIG. 10. Renaturation gel assay comparing activities present in 1 cell equivalent of X. Laeuis oocyte (Lane 1) with 10 pg (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 (Lanes 2 ,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 results of 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 this storage appears to be essentially nuclear.

However, the 5% RNase HI activity remaining in the ooplasm are very likely responsible for the reported antisense- mediated destruction of messenger RNAs. The presence of this enzyme in the cytoplasm can be the result of several phenom- enons. First, freshly synthesized enzymes on their way to the nucleus will contribute to the 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 frag- ments of RNase HI, still enzymatically active but unable to return to the nucleus. Last but not least, this distribution could simply reflect a steady-state equilibrium between proteins en-


tering the nucleus and leaving it. None of these contributions are a priori exclusive from each other, but their respective importance is hard to estimate at present. An additional natu- ral process which would increase the amount of 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 be- ginning of oogenesis, a process which takes months to provide 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 that observed in somatic cells. The conclusion is that in each of these cells the RNase HI does not seem tightly linked to chromatin and remains free to diffuse.

The apparent lack of class I1 RNase H activity in the oocyte, as inferred from assays in solution is surprising as it would implyxenopus to be an exception among the very different and phylogenetically distant organisms such as Crithidia fascicu- lata (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 has r e~umed .~ Preliminary 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 this assay. 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 characteris- tics of RNase HI1 in Krebs ascites cells (58), and for well char- acterized mammalian RNases HII.4 The finding that this polypeptide is highly concentrated in mitochondria and almost absent from the ooplasm and that its presence 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 the liquid assay is not precisely known. Our sedimentation studies suggest that it could be the result from the participation of this “cryptic RNase HII” to a larger multimeric complex, perhaps as a part of an RNA polymerase as shown for the 49-kDa subunit of yeast RNA polymerase (59). This property could also explain why the 32 kDa was not detected in the flow-through fractions of the DEAE column (data not shown). If this interpretation is correct, this would suggest that detectable RNase HI1 activity in tissues from other organisms may in fact 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 in mitochondrial nucleoprotein complexes, but its molecular mass is only 25 kDa, as determined on activity

data. P. Frank, S. Albert, C. Cazenave, J. J. and ToulmB, unpublished

Frank, P. (1986) Eu Karyonten Ribonucleasen HI. DiplomArbeit, Fakultat fur Chemie und Pharmazie, Universitat Tubingen, Germany.

gels, and its activity is readily detectable in solution with either Mg2‘ or Mn2+ (61). In vertebrates, a low abundance endonucle- ase, Endo G, has been found to possess RNase H activity in addition to DNase and RNase activities (62). It is distributed in both the nucleus and the mitochondria, acts as a 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 the mitochondria. None of these proteins has properties described for a eukaryotic class I1 RNase H. Future work on purified mitochondria from various sources should help to obtain a better understanding of the status of 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.

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Characterization and subcellular localization of ribonuclease H