Characterization of ribonuclease H activities present in two cell-free protein synthesizing systems, the wheat germ extract and the rabbit reticulocyte lysate

Biochimie (1993) 75, i 13-122

© Soci6t6 fran~:aisede biochimie et biologie mol6culaire / Elsevier, Paris

113

Characterization of ribonuclease H activities present in two cell-free protein synthesizing systems, the wheat germ extract and the rabbit reticulocyte lysate C Cazenave -~, P Frankb*, W Bi.isenb aLaboratoire de Biophysique Mol&'ulaire, INSERM CJF 90-13, Universitt; de Bordeaux H, 146, rue Lt~o-Saignat, 33076 Bordeaux, France; hLehrstuhl fiir AIIgemeine Genetik, Biologisches institut, Universitiit Tiibingen, Auf der Morgenstelle 28, D-7400 Tiibingen, Germany (Received 10 October 1992; accepted 20 November 1992)

Summary m Experimental evidence accumulated to date by several research groups indicates that antisense oligodeoxynucleotides targeted against messenger RNA (mRNA) sequences located downstream of the initiation codon fail to inhibit the translation of this mRNA unless the hybrid is c~eaved by RNase H. It has previously been shown that exogenous RNase H has to be added to rabbit reticulocyte lysate to obtain translational arrest (unless freshly prepared lysates are used). In contrast there is no need of exogenous RNase H by using wheat germ extract for translation because the level of endogenous RNase H is high enough to ensure cleavage of the hybrid formed between the antisense oligodeoxyribonucleotide and its complementary sequence on the mRNA. Surprisingly, we found that these two cell-free translation systems display similar amounts of RNase H activities when tested under standard conditions (extract diluted 500 times in the RNase H reaction mix). The RNase H activity of the rabbit reticulocyte lysate has a divalent cation requirement and sensitivity to inhibitors similar to class I ribonuclease H, whereas the activity of the wheat germ extract shows similarities to class II ribonuclease H. However, when these activities were assayed under conditions similar to those used for translation experiments, only highly reduced levels of activity were found in comparison to the standard assays. This reduction is due in part to sub-optimal ionic conditions tbr the endogenous RNase H activities in these extracts, and, for the other part, likely due to interactions with other proteins present in the lysates, In these conditions, however, the remaining activity tbund in the wheat germ extract was three times higher than the activity t'c.,und in the rabbit reticulocyte lysate, Whether this difference can by itself explain the indicated dift'erences in the two systems observed in hybrid-arrest of translation experiments remains open to discussion.

in vitro translation / rabbit reticulocyte lysate / wheat germ extract / hybrid-arrested translation / antisense oligonucleotide /

RNase H

Introduction

RNase H activity has been identified as a key factor in the success of hybrid-arrested translation with oligodeoxynucleotides (oligos) in in vitro translation assays (for review see [1,2]). In the wheat germ extract (WGE) several research groups have found that oligos hybridized to a given mRNA were efficient inhibitors of the translation of this mRNA and that this inhibitory effect was observed for oligos hybridizing upstream of the AUG initiation codon as well as for those hybridizing to the coding sequence of the mRNA [3,4]. This inhibition has been shown to be due to * Present address: Laboratoire de Biophysique Mol6culaire, INSERM CJF 90-13, Universit6 de Bordeaux l I, 146 rue L6oSaignat, 33076 Bordeaux, France.

cleavage of the mRNA in the DNA-RNA region by an endogenous RNase H activity present in the WGE. However, when similar experiments were performed in the rabbit reticulocyte lysate (RRL) significant differences in the extent of inhibition were observed depending on the site of hybridization of the oligo to the mRNA. Whereas oligos hybridizing near the cap 5' end of the mRNA were inhibitory, oligos directed to the coding sequence had no effect on translation unless exogenous RNase H from E co/i was added [3,51. The failure of oligos hybridizing to the coding sequence was interpreted as the result of the absence of endogenous RNase H activity present in the RRL [3] and of the presence of an unwinding activity associated with the translating ribosome downstream the start codon [6]. This interpretation was sustained by experiments performed with modified oligos which

!14 did not induce R N A cleavage by RNase H [7-9]. As a result, these modified oligos failed to inhibit translation in the reticulocyte lysate when they were targeted to the coding sequence (even after addition o f exogenous RNase H) as well as in the WGE. Both modified and unmodified oligos inhibit translation w h e n they hybridize to the very 5' end of the m R N A [8,9], presum a b l y because this inhibition is independent o f a RNase H activity. It s e e m s to rely on a direct competition with initiation factors or on the fact that the unwinding activity associated with the scanning 40S subunit is not as efficient as the one associated with the complete translating ribosome. Previous studies [9,10] have shown that, in fact, the R R L is not devoid o f RNase H activity but that this activity is generally too low to support hybrid-arrested translation unless fresh lysates are used (fresh lysates contain 2.5-fold more activity than c o m m e r c i a l lysates) [10]: This prompted us to characterize the RNase H activity levels present in R R L and W G E in more detail as a necessary prerequisite for assessing the p o t e n c y of chemically modified oligos to inhibit translation in both systems [9,11 l.

Materials and methods

Rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE) were purchased from Promega Biotec and stored under liquid nitrogen until use; E coli RNA polymerase, DNA t'rom calf thymus, RNase A from bovine pancreas, yeast RNA and unlabeled nucleoside triphosphates came from Boehringer Mannheim. Radioactive precursors, l aHIUTP and I.~-~PIATP, were supplied by Amersham. DNA from calf thymus was denatured by heating to 98°C for 10 rain followed by rapid cooling on ice. Ultrafiltrate of RRL was obtained by centrifugation of the lysate through a low-protein.binding type PLGC Ultrafiltration membrane (Ultrafree-MC 10000 NMWL Filter unit from Millipore). Two uitrafiltration units were filled each with 200 lal lysate and centrifuged for 90 min at 2000 g; 100 ~tl ultrafiltrate was recovered from each unit. Labeled RNA-DNA hybrids were obtained by transcription of denatured DNA from calf thymus with RNA polymerase from E c'oli using labeled NTPs.

i'~HIRNA-DNA hybrM This was prepared as follows: to a tube in which 100 laCi of [3HI-UTP were evaporated to dryness we added 0.7 ml denatured DNA from calf thymus (0.7 mg/ml), 0.25 ml 10 × transcription buffer (0.3 M Tris-HC! (pH 7.8), 0.3 M MgCI2; 1.3 M KCI: 0.01 M l~-mercaptoethanol), 62.5 lal of each NTP (0. i M) except UTP, 4.2 ktl UTP (0. I M), i.27 ml H~O and 86 l.tl RNA polymerase from E coli (IU/l.tl). After 30min incubation at 37°C the mixture was passed over a Sephadex G50 column (2.6 cm diameter x 33 cm height) equilibrated with 0.1 M NaCI (flow rate: 20 ml/h) to separate the hybrid from nonincorporated nucleotides. The fractions containing hybrid were pooled (total volume: 21 ml). Then 0.54 ml 20% SDS was added to denature the polymerase followed by 1.38 ml 1 M

KCI. The precipitated salt of potassium dodecylsulfate was removed by centrifugation for 15 rain at 15000 rpm in a Sorvall SS34 rotor. To the supematant (- 23 ml) 100 ~tl yeast RNA (12 mg/ml) was added, then 92 ml of ethanol and the sample was stored for precipitation at -20°C overnight. The precipitate was centrifuged 2 h at 4000 rpm in a Sorvall GSA rotor. Pellets were dissolved in 10 ml 0.1 M NaCI, an aliquot was counted and the solution was further diluted with 0.1 M NaC1 up to a final volume of 75 ml so that 20 H! hybrid solution counted 2200 cpm. This solution containing 5.3 pmol of hybrid/lal was aliquoted in Eppendorf tubes and stored at-20°C until use.

[32P]RNA-DNA hybrid This was prepared as follows: in an Eppendorf tube we put 25 lal 10 x buffer (0.1 M Tris (pH 7.9), 0.1 M MgCI2; 0.014 M ~-mercaptoethanol), 31 lal heat-denatured calf thymus DNA (0.7 mg/ml), 2.5 lal of each NTP (0.01 M) except UTP, 1.5 l.tl UTP (0.01 M), 0.5 lal [ot-32Pl UTP (800 Ci/mmol; 20 mCi/ml), 180 ILl H20 and 5 ~tl RNA polymerase (IU/HI). After 30 min incubation at 37°C the reaction was stopped by adding 2.5 H! SDS 20% and the hybrid was phenol extracted. The aqueous phase was brought to 0.2 M NaC! and the hybrid was precipitated with 2 volumes ethanol; after 5 rain incubation in a dry ice-ethanol bath the tube was centrifuged 10 min in an Eppendorf centrifuge, the supernatant was discarded and the pellet dried under vacuum. This pellet was redissolved in 100 lal ammonium acetate (2.5 M) and precipitated again by addition of 300 l.tl ethanol and incubation for 10 rain in a dry ice-ethanol bath. After centrifugation the pellet was redissolved in 100HI TE buffer (Tris-HCI 0.01 M (pH8.0), EDTA 0.001 M). We checked that more than 88% of the radioactivity recovered was TCA-precipitable and could be converted into TCA soluble form after digestion by E coli RNase H.

RNase it assays Fhese were performed in standard conditions as previously described [121 with minor modifications: in 1.5-ml Eppendorf tubes, we put on ice in the following order: 50 BI i0 × buffer (0.3 M Tris-HCi (pH 7.8), 0.014 M 13-mercaptoethanol, 0.1 M MgCl2 and 0.3 M (NH4)2SO4),water to adjust the final volume to 0.5 ml, extract containing enzyme, 20 Pl of the hybrid solution ( 106 pmol). The assay mixture was incubated for 10 min at 37°C, chilled on ice and 50BI yeast RNA (12 mg/mi) was added, followed by 500 BI 8% TCA. The mixture was kept on ice for i0 min, then centrifuged in an Eppendorf centrifuge for 10 min at 4°C. The supernatant was transferred to a scintillation vial and mixed with l0 ml scintillation fluid (Ecoscint A from National Diagnostics) and counted in an LKB mini-beta liquid scintillation spectrometer. All assays were performed in duplicate and the value retained was the average of the two determinations subtracted from the blank value (usually around 100cpm) determined from an assay in which the enzyme was omitted. In some assays the ionic conditions of the assay mixture were changed as indicated in the figure legends. Assays carried out under 'translation conditions' were as follows: in Eppendorf tubes we mixed on ice either 17 Ial RRL and 8 pl hybrid or 15 bti WGE, 3 I.tl O. ! M potassium acetate, 4 HI H20 and 8 BI of hybrid solution so that, in both cases, conditions were identical to the conditions we used in our translation assays [9] with the exception that aminoacids were omitted and that mRNA were replaced by the hybrid. Accordingly to the manufacturer's certificate of analysis, the

115

RRL contains 140 mM potassium acetate and 1 mM magnesium acetate, and the WGE contains 120 mM potassium acetate, 5 mM magnesium acetate, 6 mM P-mercaptoethanol and 20 mM Hepes (pH 7.6). Tubes were incubated in duplicate for the indicated times at 30°C for the RRL and at 25°C for the WGE and the reaction stopped by adding 120 ~1 H20, 50 ~1 yeast RNA (12 mg/ml) and 200 ~1 8% TCA. Tubes were then processed as described above, supematants of the tubes were pooled in scintillation vials and counted.

100

Activity gels Activity gels were performed by running classical 13% SDS polyacrylamide gels ( 14 x 11 x 0.1 cm) except that 32P-labeled RNA-DNA hybrid (= 3 105 cpm) was included in the resolving

gel before polymerisation. 5 ~1 of each extract were mixed with loading buffer containing SDS and P-mercaptoethanol, heated 5 min at 37OC,and loaded. At the end of the electrophoresis (when the tracking dye bromophenol blue reached the bottom of the gel), the part of the gel on which molecular mass markers were run in parallel with the samples was cut out and stained with Coomassie blue. The other part of the gel was processed accordingly to the procedure described by Rong and Carl [ 131 using Nonidet P 40 (NP 40) to allow renaturation of proteins inside the gel. The gel was then dried and autoradiographed overnight. Protein concentrations were determined accordingly to the procedure of Bradford [ 141,using the reagent from Biorad and bovine serum albumin as standard. These

Hybrid-arrested

translation

Experiments were performed as described previously 191

The protein samples were separated by SDS/polyucrylumidc electrophoresis und trunskrred to nitroctillulose (5 h ut 250 mA in 13 mM Nu&!Q (pH 9.9), 20% methanol). The nitrocellulose sheet was blocked with bovine serum albumin and incubated overnight with dilutions of rabbit serum (either antiserum or control serum). IgG binding was visualized by incubation with peroxidase-conjugated goat anti-[rabbit IgG(H+L)] followed by the peroxidase reaction.

Results Similar RNase H activity levels are observed in both the WGE and the RRL when tested under standard assay conditions (fig 1). In this assay, the activity found in the RRL is even a little bit higher than in the WGE. This finding was unexpected as the RNase H content was thought to be lower in RRL than in WGE, as inferred from hybrid-arrested translation assays (see Introduction). However, similar results were obtained with two other batches; the batches used were tested for hybrid-arrested translation and were found to behave in the expected manner: an oligo

4 pl

6

extract

Fig 1. Comparison of RNase H activities present in the rabbit reticulocyte lysate (squares), and in the wheat germ extract (circles) in ‘standard’ assay conditions, as described in Materials and methods, with DNA-RNA hybrid (closed symbols, solid lines) or denatured hybrid (open symbols, dotted lines).

complementary to nucleotides 113-l 29 of rabbit P-globin mRNA failed to inhibit the translation of globin in the RRL at 5 PM whereas it promoted complete specific inhibition of P-globin at 1 pM in the WGE (data not shown). The activities examined in this assay correspond truely to RNase H activities as the denatured hybrid is not degraded (fig 1), indicnting the presence of little, if any, activity corresponding to the ribonuclease H IIa in calf thymus [ 12, IS 1, The specific activity of our RRL (0.8 U/mg) was close to the one (1 U/mg) determined by Walder and Walder [lo] for RRL purchased from the same supplier (assuming that the hybrid we used is hydrolysed as efficiently as the poly(rA)mpoly(dT) they used for their studies). We found only 25% of the activity Minshull and Hunt found in their home-made WGE used for hybrid-arrested translation [3], The amount of enzyme activity of our lysate was sufficient to promote hybrid-arrested translation. The volume activity of the two extracts is nearly identical so that, theoretically, similar activities should be present in translation assays which require 17 pl RRL or 15 ~1 of WGE (see Materials and methods). However, the specific activities greatly differ due to the big difference in protein content of the two extracts, 130 mg/ml for RRL and 34 mg/ml for WGE, mostly attributable to the enormous amount of hemoglobin present in the RRL. To

116

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Fig 2. Ionic requirements of RNase H activities of cell-free translation systems. A, B. Divalent metal cation dependence of the RNase H activity of the reticulocyte lysate (A) and of the wheat germ extract (B); activity was tested for magnesium (open squares) or manganese ions (open circles). C, D. Variation of RNabe H activities from reticulocyte lysate (C) or wheat germ extract (D) with concentration of salt under 0.5 mM manganese chloride (open circles) or 10 mM magnesium chloride (closed squares). Amounts of extracts used (0.8 lal RRL or I lal WGE) were such that less than 60% of hybrid was degraded in 10 min at 37°C, thus tests were pertbrmed in the linear range of the assay. further characterize the activities present in RRL and WGE, we examined both their ionic requirements and their sensitivity to inhibitors, to distinguish between the two classes of RNase H activities present in all higher eukaryotes examined so far [ 161. As illustrated in figure 2, big differences are found between the two systems. Whereas the activity present

in the RRL is stimulated equally well by magnesium (optimum = 5-10 mM Mg2+) or manganese (optimum 0.5-0.6 mM Mn2+) the activity of the wheat germ extract is almost dependent upon added Mg2+ (optimum 10 raM) as the activity in the presence of Mn2+ (optimum 0.2-0.4 mM determined in an independent experiment) is greatly reduced (fig 2A, B).

117 120

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Fig 3. Sensitivity of RNase H activity from the reticulocyte lysate (solid lines) or wheat germ extract (dotted lines) to inhibition by N-ethyl maleimide. Assays were performed under 'standard condition' with either 10mM MgCI2 (squares) or 0.5 mM MnCI2 (circles), using 1 ILtlextract per test. Activities present in the two systems can also be distinguished by their sensitivities to salt (fig 2C, D). Whereas the activity in the RRL is highly sensitive to added salt (complete inhibition at 100 mM (NH4)2SO4 with 10 mM MgCI2) the activity present in the WGE is only moderately inhibited. As class I RNases H are equally active under Mg2, or Mn2+ and are sensitive to added salt, and class 11 RNases H are stimulated by Mg2+ and not by Mn2, and relatively insensitive to salt [12,16], our results suggest that the predominant activity present in the RRL is a class I RNase H, and that the one found in ~ G E is a class II RNase H. This interpretation is supported by the sensitivity of these activities to N-ethyl maleimide (NEM) (fig 3). It is known that class II RNase H is highly sensitive to NEM [15] and that class I is insensitive to NEM in the presence of manganese [12]. Whereas the Mn2*dependent activity of the RRL is only moderately sensitive to NEM even at high concentration, the Mg2+-dependent activity of WGE is highly sensitive to low concentrations of NEM. The Mg2+-dependent activity of the RNase H in the RRL is also inhibited by NEM but the concentrations needed to achieve the same extent of inhibition seen for the WGE are 2-5 times higher. The finding that the sensitivity to NEM

depends on the metal used in the incubation mixture. is puzzling. This observation has been already reported for calf thymus RNase H I [ 12]. One plausible hypothesis is that the local folding of the protein, which determines accessibility of NEM to cysteine residues necessary for activity, is dependent on the divalent cation used. Another possibility is that the RNase H may interact with another protein in a divalent cation-dependent manner, and that this association between proteins dictates the sensitivity of the nuclease to NEM. Similar differences between the enzymes of the RRL and the WGE are found when we examined the sensitivity of the enzymes to added Mn2+ ions in the presence of l0 mM Mg 2+ (fig 4). The manganese sensitivity is a characteristic feature of class II enzymes [15]. Whereas the RNase H activity of the RRL is only reduced by 25%, the activity of the WGE is inhibited by more than 85%. Taken together, these results can be interpreted by the presence of a class I RNase H in the RRL which may account for about 80% of the activity and the predominant presence of a class II RNase H in the WGE. We attempted to further characterize the RNases H in both extracts by performing activity gels (fig 5) and immunoblots with antibodies raised against purified RNase H I and RNase H lib from calf thymus (fig 6). Whereas no activity is detected in the activity gel for the RRL, an activity is present in the WGE for a poly-

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118 peptide of about 32 kDa. Such activity is seen for purified RNase H II from Krebs II ascites cells [ 17], RNase H lib from calf thymus and from human placenta (P Frank and C Cazenave, unpublished results). However, such an activity is also detected in preparations of RNase H I and seems to correspond to a proteolytic fragment of the enzyme [ 13]. Immunoblots probed with an antibody raised against calf thymus RNase H I clearly indicate the presence of a cross-reacting band corresponding to a polypeptide of about 52 kDa in the RRL (fig 6). Such a polypeptide is also detected in freshly prepared extracts from calf thymus but seems to be rapidly converted into smaller polypeptides of 32 kDa and 21 kDa due to proteolytic cleavage in the early steps of the purification [181. As it appears now that the native class ! enzyme protein is of higher molecular mass (about 68-70 kDa) [131, this would suggest that this 52-kDa polypeptide could correspond to a proteolytic product rather than to the native enzyme protein. The 52-kDa polypeptide, however, is not detected in activity gels, most probably because large proteins are less easily renaturable than smaller ones [ 13 I.

RRL

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94-67-43--

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Fig 5, Activity gel run with rabbit reticulocyte lysate or wheat germ extract, as described in Materials and methods. A clear band corresponding to = 32 kDa in the WGE track indicates RNase H activity. Bands darker than the background level correspond to 'hybrid-binding' proteins [23].

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Fig 6. Immunoblot analysis of RRL and WGE: 100 lag of RRL (lanes I in the first panel) and 100 lag WGE (lanes 2 in the second panel) have been separated on a 10% SDS/polyacrylamide gel and transferred to nitrocellulose as described in Materials and methods. For immunoblot analysis (see Materials and methods) the following rabbit sera have been used in a I :!000 dilution; o~ H l = antiserum raised against purified bovine ribonuclease H I [20]; ~H lib = antiserum raised against purified bovine ribonuclease H lib [19] and C = control serum of a non-immunized rabbit, respectively.

in the WGE the picture is not as clear-cut: prominent polypeptides with RNase H I antigenicity are seen in the M~ range of 20 kDa and at least two faint ones in the M, range of 60 kDa. It is not clear to us whether they correspond to a minor class I enzyme activity in the WGE. The antibody raised against RNase H lib does not detect any polypeptide in the RRL. However, it decorates a distinct polypeptide with a M, of around 31 kDa in the WGE. This band could correspond to the polypeptide of about 32 kDa seen in activity gels. In summary, class I antigenicity is seen in RRL, and eventually also in WGE, class II antigenicity only in WGE. All these findings are at least compatible with the former notion that RRL displays predominantly a class I ribonuclease H activity, and W G E predominantly a class II ribonuclease H activity. The RNase H acthity levels found in both extracts are similar even if ene compares them in their respective optimal conditions (5 mM MgCI2, 5 m M ammonium sulfate for the RRL enzyme, l0 mM MgCI2, l0 m M ammonium sulfate for the W G E enzyme). However, a completely different picture emerges under conditions used for translation where ionic conditions should be different and the protein concen-

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F,~g7. RNase H activity of RRL (squares) and WGE (circles) under 'translation conditions', peffonned as described in Materials and methods, tration is very high. In our standard assay we used 1-2 I.tl of the extract diluted in 500 I.tl final volume whereas under 'translation conditions' we used 17 lal RRL in 25 pl final volume and 15 I.tl WGE in a 30-l.ti final volume. Thus, in the RRL translation assay the protein concentration is 340 times higher than in the standard assay. Similarly, in the WGE translation assay the protein concentration is 250 times higher than in the standard assay. We decided to test the levels of hybrid hydrolysis under conditions identical to those used in translation assays (fig 7). This ¢xperiment displayed a significant difference in the rate of degradation of the hybrid between the two systems, the WGE being two times more active than the RRL despite the lower tempera-. ture (25°C) used for the WGE compared to the temperature (30°C) used for the RRL. In both systems, however, the RNase H activity is greatly reduced in comparison to the activities monitored under standard assay conditions: the activity observed in 'translation conditions' is only 1.2% of the activity observed under 'standard conditions' for RRL and 6% for WGE. We decided to find out why the RNase H activity was so strongly inhibited in the RRL under 'translation conditions'. We first explored the possibility that the apparently low level of activity seen in the RRL was due to an unwindase activity present in the lysate which could have melted the DNA-RNA hybrid and rendered the RNA moiety resistant to RNase H digestion. This possibility was ruled out by the experiment

presented in figure 8. This experiment compared the amount of hybrid hydrolyzed by the RRL with or without addition of RNase A for 2 min before arresting the reaction by addition of carder RNA and trichloracetic acid (TCA). This treatment with RNase A was sufficient to completely digest denatured hybrid under 'translation conditions ". The two curves are not coincident because about 20% of our hybrid preparation is sensitive to RNase A. As the slope of the line with RNase A treatment is not significantly much higher than the one without RNase A treatment, a melting activity, if present, cannot by itself explain the inhibition of RNase H activity observed in 'translation conditions' compared to 'standard conditions' in the RRL. We then decided to check if the ionic conditions could be responsible for the very low activity found under 'translation conditions'. We first observed that addition of 4 mM Mg 2÷ increased 1.8-fold the RNase H activity in translation c~nditions. This led us to conclude that ionic conditions were not optimal for the activity under these conditions. We then compared the activity of 1 lal RRL diluted either with 17 lal of three distinct buffers as indicated or 17 ktl ultrafiltrate of RRL (that is RRL devoid of proteins larger than 10 kDa) and 8 gl hybrid. Buffers used were either 20 mM Hepes (pH 7.6), 140 mM K

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Fig9. Comparison of the RNase H activity of I ~tl RRL diluted either in 17 lal ultrafiltrate or 17 ~tl of different buffers. To the diluted enzyme solutions, 8 lal of hybrid was added and the reaction incubated for 10 min at 30°C and processed as described for the assays under 'translation conditions' (see Materials and methods). Tris buffer, 30mM Tris-HC! (pH7.8), 30 mM (NH4)2SO4, 10mM MgCI,, 0.01% 13-mercaptoethanol. With this buffer 36 pmol hybrid were hydrolysed. acetate, I mM Mg acetate (intended to mimic conditions thought to be present in the lysate, according to the specifications of the manufacturer) or the same buffer containing 5 mM Mg acetate (corresponding to the concentration present in the WGE, according to the manufacturer). Results were compared to those obtained with the buffer used tbr testing the calf thymus enzyme. As summarized in figure 9 the activity tbund in the uitrafiltrate (which represents truely the ionic conditions in the lysate) is very similar to the activity observed in the buffer containing 1 mM Mg acetate; this activity is only 1/4 of the activity found in the 'calf thymus' buffer. Thus due to the non-optimal ionic conditions in the reticulocyte lysate, one has to expect a reduction of enzyme activity in the range of 70-75% in comparison to optimal conditions. A likely explanation for the much higher inhibition actually observed (about 99%) is that in addition to the suboptimal ionic conditions inhibition by direct protein-protein interactions may occur in the highly concentrated protein solution of the lysate.

Discussion Our study indicates that, in contrast to the general assumption, RNase H activity levels are similar in

both the wheat germ extract and the rabbit reticulocyte lysate. Moreover, it shows that the predominant RNase H of the RRL is a class I RNase H whereas the predominant RNase H of the WGE is a class II RNase H. lmmunoblots and activity gels are in agreement with this conclusion. Immunoblots show a unique cross-reacting polypeptide of about 52 kDa in RRL, in accordance with previous observations on RNase H I of calf thymus extracts. It should be noted that neither activity gels nor immunoblots detect shorter polypeptides of about 32 kDa indicating that little, if any, active proteolytic products of RNase H I are present in the lysate. On the contrary, the activity gel run with WGE clearly shows all activity corresponding to a protein of about 32 kDa which could be the class II enzyme, as we observed the same picture for RNase H lib from calf thymus or human placenta. We cannot exclude, however, that it could correspond to a proteolytic fragment of H I which could represent the minor RNase H activity present in WGE which can be activated with manganese ions. The fact that the predominant enzyme found in the WGE is a class II ribonuclease H was a big surprise, because it has been asserted previously that class II enzymes were exonucleases, a conclusion inferred from the fact that RNase H lib was unable to relax supercoiled plasmids containing stretches of RNA !191, whereas class I ribonucleases H does [20]. The mode of action o~ antisense oligonucleotides by inducing RNase H cleavage of the mRNA implies that this RNase H activity is an endonuclease. This led us to reexamine the exo-versus endo-nuclease activity of class 11 ribonuclease H from calf thymus. We found indeed that the enzyme is able to cleave mRNA hybridized to an oligonucleotide, so that we now think that the failure of RNase H lib to cleave plasmids is rather due to supercoiling and not due to a lack of endonuclease activity (Cazenave et al, unpublished results). This study indicates that under conditions used for translation both activities are highly reduced in comparison with the activities observed under standard conditions. A study focused on RRL clearly indicates that ionic conditions are sub-optimal for activity (Mg acetate concentration too low and K acetate presumably too high). However, additional factors, as for example the association of the enzyme with other proteins in the proteinaceous concentrate of the iysate, may account for the actual inhibition of activity. However, even under 'translation conditions' the relative activities between WGE and RRL only differ by a factor of two (or three if activities are compared at the same temperature) whereas hybrid-arrested translation experiments indicate a nearly all or nothing difference between the two systems. We have used an artificial DNA-RNA hybrid; this may not reflect the exact situation when a true mRNA is used. This

121 m R N A will be covered by ribosomes and embedded in a cloud of proteins involved in translation (initiation and elongation factors, tRNA synthetases, etc.) which may somehow prevent access of other proteins. In that context, smaller RNases H could be favoured for access to the short mRNA-oligonucleotide hybrid, as for example RNase H lib ( - 3 2 k Da) or E coli (= 17kDa) versus RNase H I ( 5 2 k D a or even 68 kDa). In addition, as illustrated by the darker bands observed in the activity gels, many other proteins may bind to the hybrid and may therefore compete with the RNase H or restrict the length of hybrid available for cleavage. In this situation, the activity of smaller RNases H could be favored in comparison with larger ones.

Conclusion One major conclusion of this study is that one cannot infer from the presence of high RNase H levels in one compartment that antisense oligonucleotides will disrupt any RNA targeted inside this compartment. For example, the fact that the nucleus contains the major part of RNase H of the cell ([19] and Cazenave et al, unpublished results) does not imply that antisense oligos will be more efficient when targeted against nuclear targets than cytoplasmic ones. Secondly, the size of the RNase H involved could well play a role and their efficiency for antisense cleavage of RNA may be inversely proportional to their size. In that context, minor proteolytic degradation products of RNases H, with conserved enzyme activity, could play a bigger role in antisense mechanisms than the larger native proteins. Along the same line, the relative amounts of RNase H 1 and RNase H 1I activities have been shown to vary accordingly to the physiological status of the cell (see [21] for review) as RNase H I seems to correlate with DNA replication and RNase H II with RNA transcription [22]. Therefore, the efficiency of antisense oligos in inducing RNase H cleavage of RNAs in living cells may vary accordingly with the relative amounts of RNase H of both classes and so with the physiological state of the cell, irrespective of other parameters (oligonucleotide penetration, compartimentalisation, turn-over, etc.) which are also expected to be dependent on the status (resting, dividing, differentiating, etc) of the cell.

Acknowledgments This work was initiated when C Cazenave was at the Laboratoire de Biophysique INSERM U201, CNRS UA 481, Museum National d'Histoire Naturelle, Paris, France. Professor Claude H61~ne, director of this laboratory, is gratefully acknowledged for providing lab space and support at that time.

Authors want to thank Dr Claudille Boiziau (Bordeaux) for performing the hybrid-arrested translation experiments, Dr Jean-Jacques Toulm6 (Bordeaux) for suggesting to test the presence of an unwindase activity in the rabbit reticulocyte lysate, Dr Heiner Vonwirth (Tiibingen) for providing the antibody against calf thymus ribonuclease H lib and Mrs Kati Pierozzi for carefully typing the manuscript.

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