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Conceived and designed the experiments: HP AW JML YA. Performed the experiments: AW JML MC YA. Analyzed the data: HP AW MC YA. Contributed reagents/materials/analysis tools: HP AW JML. Wrote the paper: HP JML YA.
We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE) or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE) from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i) the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii) the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3′dAMP), but sensitive to cordycepin 5′-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii) the RNA polymerization is not a 3′ end labelling and that iv) the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp).
Post-transcriptional regulations modulating mRNA stabilities represent important mechanistic steps in the regulation of eukaryotic gene activity. Indeed, it is clearly assumed that half of all changes in the amounts of mRNA is attributed to altered rates of decay , . Since 20 years, distinct programs of mRNA decay have been described as post-transcriptional networks controlled by proteins and/or non-coding RNAs which bind to specific sequences or structural elements in the regulated RNAs , . Among them, post-transcriptional gene silencing or RNA silencing first discovered in plants and fungi, was reported for a number of animals and protozoa as RNA interference (RNAi) , . RNAi is the process whereby double-stranded RNA (dsRNA) induces the homology-dependent degradation of cognate mRNA. This dsRNA is converted into many 21–25 nt short interfering RNA (siRNA) fragments that are further processed by an RNA dependent RNA polymerase (RdRp) activity resulting in the production of secundary siRNA , . Soluble recombinant RdRp proteins were purified from tomato and Neurospora crassa , . They were characterized as RNA template-dependent RNA polymerases in vitro and biochemical and genetic experiments have established that RdRp plays a critical role in amplifying the RNAi effect , . Up to date, RdRp homolog has only been detected in the genomes of plants, fungi, yeast and nematode , ,  but not in the genomes of Drosophila and vertebrates. However, post-transcriptional suppression of gene expression by siRNA was established in oocytes and preimplantation embryos of mice  as well as in Xenopus embryos . A coupling between RNA amplification due to an RNA dependent RNA synthesis and RNA degradation was evidenced in Drosophila RNAi where repeated cycles of dsRNA synthesis and concomitant siRNA/primer production result in mRNA degradation , . Overall RNAi is conserved in species where no RdRp gene is detected. As a matter of fact, recently, an RdRp activity involved in RNAi and transposon silencing was identified in Drosophila . Moreover, an RdRP activity was isolated from human cells and consists of the ribonucleoprotein complex of telomerase reverse transcriptase catalytic subunit (TERT) and of the RNA component of mitochondrial RNA processing endoribonuclease (RMRP) .
Using an in vivo heterologous system to study RNA stability at the post-transcriptional level, we previously reported a coupling between RNA amplification and accelerated degradation of in vitro synthesized Xenopus transcripts injected into axolotl oocytes, fertilized or unfertilized (UFE) eggs . After injection, the exogenous RNAs are not continuously degraded over time but are detected with amounts sometimes higher than the injected amounts. The phenomenon occurs with a variety of exogenous sense and antisense substrates. We also established that most of the molecules after injection have the same polarity as the initially injected RNA. Cordycepin 5′-triphosphate prevents increases in RNA levels indicating the involvement of an RNA synthesis . Moreover, we detected the presence of trace amounts of complementary RNA (cRNA), in agreement with the involvement of an RdRp in vivo . Similar results were obtained in vivo after injection of axolotl RNA into Xenopus fertilized eggs, suggesting a phylogenetically conserved mechanism.
In order to further investigate the molecular mechanism of this in vivo post-transcriptional process, we intended to use cell-free extracts. In the present study, we develop for the first time low speed extracts (LSE) from axolotl UFE, according to a protocol that minimizes dilution as previously published for Xenopus laevis . Such extracts from Xenopus UFE have provided the first non viral cell-free system able to faithfully reproduce nuclear synthesis events, from the replication of DNA to the assembly and reconstitution of a functional nucleus ,  and either low- or high- speed extracts can be obtained, depending on the mechanism to be analyzed or the question to be addressed , . After characterization of the catalytic properties of the axolotl extracts towards a single stranded M13 DNA dependent DNA polymerization activity, we tested the axolotl and Xenopus extracts in an RNA polymerization assay. We show that the amphibian cell-free systems incorporate radioactive ribonucleotide precursors into discrete RNA polymers, indicating that axolotl and Xenopus extracts contain all the components necessary to polymerize RNA. When large amounts of an exogenous RNA are added to the extracts, the labelling of a single RNA band similar in size to that of the exogenous RNA is detected. This radioactive labelling does not depend on the free 3′OH extremity of the added RNA and RNA polymerization is not due to terminal labelling of the 3′ end of the RNA. Analysis of the radiolabelled RNA sensitivity to RNase A shows that the RNA produced is single stranded. These results support the hypothesis of an RNA dependent RNA neosynthesis in the axolotl and Xenopus mitotic extracts.
The protocol of animal handling and treatment was performed in accordance with the guidelines of the animal ethics committee of the Ministère de l′Agriculture of France.
Axolotl UFE were obtained from females maintained at 18°C in our laboratory, stimulated 24 h earlier by subcutaneous injection (700 UI) of human chorionic gonadotrophin (hCG; Chorulon, Intervet, France) and kept overnight in 1X HSB (15 mM Tris pH 7.6, 110 mM NaCl, 2 mM KCl, 1 mM MgSO4, 0.5 mM Na2HPO4, 2 mM NaHCO3) at room temperature. An axolotl female lays between 800 to 1000 eggs (2.3 mm in diameter). Laid eggs were collected, quickly rinsed with 0.2X HSB brought to pH 8 by addition of NaOH 10N and completely dejellied by gently swirling at room temperature in 3% L-cysteine-hydrochloride, 0.1% papain in 0.2X HSB pH 8. Dejellied eggs were rinsed at least 5 times in 100 mL 0.2X HSB, EGTA 1 mM pH 7.8 per mL of eggs (EGTA : Ethylene Glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), sorted under a dissecting microscope to remove all damaged or abnormal eggs and eggs were immediately used for preparation of cell-free extracts. Xenopus UFE were obtained as previously described , .
Axolotl eggs were first incubated during 5 min in 25 mL cold 1X CSF, 5 mM EGTA buffer extract (2X CSF : 100 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2, 10 mM potassium Hepes pH 7 made by addition of KOH, 50 mM sucrose) then transferred 5 min in 25 mL of 1X CSF, 5 mM EGTA buffer extract containing 10 µg/mL of each protease inhibitors (leupeptin, pepstatin and aprotinin; Sigma). UFE were then transferred in 2.2 mL eppendorfs previously cooled on ice. Excess buffer was removed, leaving only interstitial buffer between packed eggs. After centrifugation at 20 000 g during 50 min (16°C), the cytosolic supernatant was collected, excluding the lipid overlayer, the yolk pellet and the particulate grey interface. The supernatant was collected and Cytochalasin B (10 mg/mL, Sigma) was added to a final concentration of 10 µg/mL. All subsequent manipulations were carried out at 4°C. Cytosolic supernatant was cleared by centrifugation at 20 000 g during 50 min and the soluble phase was collected by puncturing the side of the tube with a 20-gauge needle inserted into a 1 to 5 mL syringue then stored on ice. Protease inhibitors at final concentration of 10 mg/mL each, Energy Mix (1/20 volume) and 5% glycerol were added (Energy Mix 20X : 200 µg/mL Creatine Kinase, 200 mM Creatine Phosphate, 20 mM ATP, 20 mM MgCl2, 2 mM EGTA). At this point, Cytochalasin B was added (1 µL at 10 mg/mL per mL of supernatant). After gentle mixing, the clear extracts obtained were stored at −80°C in 50 µL aliquots rapidly frozen in liquid nitrogen. One milliliter of extract was prepared from 500 axolotl UFE. Xenopus low speed extracts (LSE) were prepared as previously described . In axolotl and Xenopus LSE, the final concentration of non-yolk proteins was determined by Bradford dosage according to Maniatis and Sambrook  and estimated to 24 µg or 26 µg for respectively one axolotl or Xenopus UFE. These values (25 µg) are in perfect accordance with those found for non yolk proteins in mature Xenopus stade VI oocytes . The concentration of total RNA in each extract was determined by LiCl precipitation, phenol/chloroform-chloroform extractions and by absorbance at 260 nm (A260) and estimated to respectively 1 and 1.5 µg per one equivalent UFE in axolotl and Xenopus extract.
All the plasmids used as templates in DNA polymerization experiments (single-stranded or double-stranded (7246 base pairs) M13mp18 circular plasmid and double-stranded (2464 base pairs) circular pSP73 plasmid DNA) were purchased at Biolabs as RNase-free grade products and resuspended in the manufacturer's buffer Tris-HCl 10 mM pH 7.5, EDTA 1 mM. Preparation of Xenopus demembranated sperm nuclei was as previously described . Capped synthetic RNA templates were generated from linearized plasmids using an in vitro transcription kit according to the manufacturer's instructions (Ambion Inc., Austin, TX). The Xenopus pGbORFA65 construct was used to synthesize globin RNA . This plasmid contained the 5′UTR, the entire open reading frame and 20 nucleotides of the 3′UTR of the β-globin gene, followed by a poly(A) track of 65 adenines (A). Poly(A)− (without the 65 A; 0.555 kb) and poly(A)+ (with the 65 A; 0.620 kb) sense RNA were obtained by linearization of the plasmid respectively with BamHI or EcoRI (Biolabs) then transcription from T7 promoter of the Bluescript vector. C-myc Xenopus RNA was produced in vitro using T7 RNA polymerase and the template pXLmyc  digested with HindIII to generate the sense 2.2 kb full-lenght transcript. The mouse PKCζ RNA was obtained by linearization of the pBluescriptKS plasmid (gift of S. Louvet) by SalI or XbaI (Biolabs) and transcription from respectively the T3 or T7 promoter of the vector generating sense 534b SalI or antisense 540b transcripts. After the transcription reaction, removal of cDNA template was done by addition of RNase-free DNase (2 U) for 30 min at 37°C. All transcripts were resuspended in water. RNA concentrations were determined by absorbance at 260 nm (A260). The standard reaction for addition of cordycepin (3′-deoxy AMP) at the 3′OH ends of in vitro transcripts was done according to the poly(A) tailing kit procedure from Ambion (Ref : AM1350) except that cordycepin 5′-triphosphate was used instead of ATP. Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2, 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C. To check the inability of these 3′OH-modified RNAs to be elongated by any radiolabelled ribonucleotide, 100 µg of in vitro Xenopus globin or c-myc transcripts modified at their 3′OH-end (globin 3′H or myc 3′H) or not (globin 3′OH or myc 3′OH) were incubated in a standard tailing reaction (Ambion) containing 1 mM ATP, RNase Inhibitor (30 U), [α–32P] rATP (0.3 µM, 10 mCi, Perkin Elmer) and E. Coli poly(A) polymerase (12 U) in a final 200 µL reaction during 30 min at 22°C. The size and quality of the different DNA or RNA templates were respectively controlled on a native agarose gel (DNA) or on a formaldehyde gel (RNA) then by electrophoretic migration. RNase A (DNase and protease free) was purchased from Boehringer Mannheim, DNase RQ1 from Promega, α-amanitin, cordycepin and cordycepin 5′-triphosphate from Sigma and RiboRuler RNA ladders from Fermentas.
A standard in vitro reaction for a DNA polymerization assay was as follows. Aliquots of frozen extracts were thawed at room temperature immediately before use. To 44 µL of egg extract, 2.5 µL of the 20X ATP-regenerating system (Energy mix) were added to give a final concentration (1X) of 10 mM phosphocreatine (Sigma), 10 mg/mL creatine phosphokinase (Sigma), 1 mM ATP and 1 mM MgCl2. Samples were then supplemented with 1 µL of DNA (100 ng of single- or double-stranded DNA or Xenopus sperm nuclei equivalent to 25,000 demembranated sperm nuclei, 1 µL of [α–32P] dCTP or [α–32P] dATP (0.3 µM, 10 mCi) and 0.5 µL of cycloheximide (20 mg/µL, Sigma) or water to obtain a final volume of 50 µL. For a standard in vitro RNA polymerization reaction, 2.5 µL of the 20X ATP-regenerating Energy mix system were added to 42 µL of egg extract supplemented with 1 µL RNasin inhibitor (10 U, Invitrogen), 2 µL of RNA in vitro template (500 ng), 1 µL (0.3 µM, 10 mCi) of [α–32P] CTP or [α–32P] UTP and 2 µL containing drugs at the desired concentrations (cordycepin, cordycepin 5′-triphosphate or α-amanitin) to obtain a final volume of 50 µL. For all the reactions, immediately after gentle homogenization, the addition of the radiolabelled precursor defined the t=0 of the kinetics performed at 20°C, unless otherwise mentionned. At t=0, five microlitters (1/10 of the initial reaction) was added to 45 µL of Stop mix solution (40 mM EDTA, 1% SDS : Sodium Dodecyl Sulfate) then spotted on a Whatmann GF/C glass filter to determine the total radioactivity of the extract (input). At each time of the kinetics, 1/10 of the reaction was withdrawn and added to 45 µL of Stop mix solution. At the end of the kinetics, each Stop mix solution containing the samples was incubated with 2 µL of Proteinase K (10 mg/mL, Fermentas) during 2 hr at 55°C, then the totality of the reaction was directly spotted on a Whatmann GF/C glass filter. Then filters were TCA (Trichloroacetic Acid) precipitated first at least 3 hr in two batches of cold 5% TCA, 2% PPNA solution (PPNA : tetrasodium diphosphate) (20 mL per filter) and three times two hours in cold 5% TCA then washed twice in ethanol 95%, dried and counting of the incorporated TCA precipitable radioactivity was done in 5 mL of Liquifluor (Amersham). For RNA polymerization assays, an RNase A treatment (Fermentas) was added to the standard procedure : 5 µL of the reaction were incubated with 1 µL of RNase A (10 µg) and water (14 µL) during 1 hr at 37°C.
For DNA agarose gel analyses, after the polymerization assays using incubations with the amphibian extracts, some samples were treated during 1 hr at 55°C with Proteinase K (10 U/assay), then phenol/chloroform and chloroform (V/V) treated before ethanol precipitation and totally loaded in an 1% agarose native gel before electrophoresis in TEA 1X buffer as described by Maniatis and Sambrook . After transfer to Hybond N+ membrane (Amersham, Orsay, France), the filters were washed twice in 2X saline sodium citrate (SSC), pre-hybridized at 65°C in Church buffer , hybridized to a radioactive random primed M13 probe at 65°C then washed at this temperature in 0.2X SSC, 0.1% SDS and exposed to a screen before PhosphoImager analysis (Amersham, Molecular Dynamics). For RNA gel analyses, after the polymerization assays, total RNA from each reaction was extracted by the lithium chloride (LiCl) method . Total RNA was then heated 8 min at 65°C and separated by electrophoresis in 1.3% agarose/formaldehyde/MOPS (3-(N-Morpholino) propanesulfonic acid) gels that were first ethidium bromide stained and photographed under UV light. Alternatively, total RNA was separated in 4% acrylamide-bisacrylamide (29 :1) - 8 M urea (TBE 1X) gels that were stained by Stains-all according to the manufacturer (Fluka) and photographed. Agarose and acrylamide gels were then dried and exposed to a screen before PhosphoImager analysis.
Either total RNA extracted from an RNA polymerization sample using axolotl LSE (48 µl) and an exogenous synthetic transcript (24 µg) or two combinations (10 µg) of in vitro transcripts (5 or 10 µg each) were heat-denatured 10 min at 85°C in 80% deionized formamide, 40 mM PIPES pH 6.7, 0.4 M NaCl, 1 mM EDTA (final RNA concentrations : 150 µg/mL). Rehybridization was performed overnight at 65°C. Then the RNA solutions as well as 10X RNase A digestion buffer (3 M NaCL, 0.1 M Tris-HCl pH 8.5, 50 mM EDTA) were diluted 10 fold in a common mix to proceed to RNase A (Fermentas) digestion for 1 hr at 37°C (final RNase A concentrations : 0 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL and 1 µg/mL). Digestions were stopped by incubation with proteinase K (2 mg mL−1), 8% SDS during 15 min at 37°C. After phenol/chloroform extraction, RNAs were ethanol precipitated in the presence of glycogen (50 µg/mL; Fermentas) and dissolved in 80% deionized formamide, 50 mM Tris-HCl pH 8.3, 1 mM EDTA, 1% bromophenol blue, 0.1% xylene cyanol. RNAs (corresponding to respectively 1/8 of the RNA polymerization sample or 1/5 of the RNA combinations) were electrophorezed on 8 M-urea 4% polyacrylamide gel for 75 or 30 min at 150 Volts in TBE buffer (0.89 M Tris, 0.88 M Borate, 10 mM EDTA pH 8).
Assuming that it was the first time that axolotl extracts were carried out, we evaluated the quality of this new in vitro system according to procedures used for Xenopus LSE . We tested the ability of axolotl LSE to catalyze DNA dependent DNA polymerization. Demembranated Xenopus sperm nuclei containing 100 ng DNA were incubated with axolotl or Xenopus extracts in reactions using [α–32P] dCTP as well as Ca++ addition or not. As expected, [32P] dCMP incorporation into TCA precipitable material was Ca++ dependent in Xenopus LSE (Fig. 1A, lanes 1–3) . However, no [α–32P] incorporation was registered in axolotl LSE (Fig. 1A, lanes 4–6). No incorporation as well was observed with double-stranded (DS) plasmid DNA used as template (data not shown) which has already been reported for Xenopus LSE . We then tested the single-stranded (SS) M13 template and figure 1A shows that it is an efficient substrate for dCMP incorporation using axolotl LSE (lanes 7 to 9). When following the incubations, the samples were DNase treated prior to TCA precipitations, no radioactivity was counted above the background defined by a sample where no DNA was added (data not shown). We thus considered that the TCA precipitated material was indeed DNA polymers. Moreover, when visualizing the DNA polymerase reaction products as well as appropriate SS or DS DNA controls in an electrophoresis agarose gel, we observed that following a 2 hr incubation, SS M13 was totally converted into different forms of DS M13 (Fig. 1B).
Overall, these results show that mitotic axolotl LSE represents a successfull eukaryotic system that efficiently converts circular single-stranded M13 DNA to double-stranded DNA.
We then tested the ability of five axolotl extracts to incorporate [α–32P] CMP into TCA precipitable material during polymerization assays in the presence of 500 ng of exogenous RNA (Fig. 2). Kinetics over a 2 hr incubation period indicated that [α–32P] CMP was indeed incorporated into TCA precipitable material since cpm registered at all the time-points of the kinetics were over the background cpm registered at t=0, reaching a mean 3 fold above background at 2 hr (Fig. 2A).
Although the amount of incorporated cpm was variable from one point to the other during the kinetics and from one extract to the other, longer incubation times up to 24 hr confirmed [α–32P] incorporation with cpm at 6 hr up to 10 fold above the background (data not shown). The use of the same extract in 7 independent experiments (Fig. 2B) did not allow us to reduce the variability in the incorporation rates with a standard deviation corresponding to 30% of the background value and 18% after 4 hr incubation. However, it clearly shows a mean increase of the incorporated cpm at 4 hr consisting in 6.5 fold above the background. Moreover, we observed that addition of 500 ng exogenous RNA had no effect on the total amount of cpm incorporated after 4 hr incubation. To further characterize the reaction products, we introduced a nucleasic treatment following a 4 hr incubation period with the axolotl extract and before the TCA precipitation step. No incorporation at all was detected when samples were treated with RNase A prior to TCA precipitation, indicating that CMP is incorporated into an RNA polymer. This absence of incorporation was not due to the modification of the protocol since the incorporation obtained in a sibling experiment that omitted RNase A addition but included a one hour incubation period at 37°C exhibited only a low decrease in the TCA precipitable cpm (Fig. 2C). Similar results were obtained for Xenopus LSE analyzed in parallel (Fig. S1). In summary, although incorporation rates were low, these experiments indicate that axolotl and Xenopus extracts contain all the components necessary to catalyze RNA synthesis. We therefore further characterized this RNA synthesis through visualization of the incubation products separated by electrophoresis in a denaturating agarose gel.
Aliquots of a standard RNA polymerisation reaction without any exogenous RNA were processed at t=0, t=1 hr and t=4 hr by LiCl method then analyzed by agarose gel electrophoresis and autoradiography. As shown in a representative experiment (Fig. 3), low signals were detected following a 4 hr incubation in the presence of radioactive CTP or UTP (lane 4), with two major bands migrating at 1.8–1.9 and 2.7 kb and with 6–7 minor discrete bands ranging from 0.8 to 1.4 kb. In this experiment, the intensity of all bands slightly increased between 1 hr (lane 2) and 4 hr (lane 3). All the signals disappeared after an RNase A treatment (lane 5) but were insensitive to a DNase treatment (lane 8), indicating that these radioactive bands corresponded to RNA. The intensity of the signals detected in the extract incubated in the presence of α-amanitin, a potent inhibitor of RNA polymerases II and III , , was similar to those obtained without the drug (compare lanes 6 and 3). This suggests that the radioactive labelling corresponds to an enzymatic activity controlled by a mechanism independent of RNA Pol II and III activities. Discrete bands were observed in the Xenopus LSE mitotic extract incubated during 4 hr with CTP as radioactive precursor (lanes 10–11). These patterns of discrete bands detected in independent experiments performed from different axolotl and Xenopus extracts were not always as clearly observed as in figures 3 and and44 (Fig. 5). However, these results overall confirm that amphibian extracts incorporate ribonucleotide precursors in RNase but not DNase sensitive products, i.e. RNA molecules, and indicate that the RNA polymerization is catalyzed by an RNA polymerase activity different from RNA pol II and pol III.
Interestingly, our preceeding experiment showed that the radiolabelled products were clearly resolved into few discrete bands. One of them corresponding to the 1.8–1.9 kb band comigrates with the 18S rRNA which represents an important endogenous amount of RNA in the extract. This observation led us to study the impact of the addition of a large amount of an exogenous RNA during RNA polymerization with the axolotl extract. Since no radiolabelled bands were detectable with sizes between 2 and 2.7 kb or smaller than 0.8 kb, we choose to design a 0.55 kb and 0.62 kb (Fig. 4) as well as 2.3 kb (Fig. 5) synthetic exogenous RNA for further analysis. We first used ten microgrammes of the in vitro synthesized Xenopus 3′UTR poly(A)− globin RNA (555 bases). This amount of exogenous RNA was equivalent to 1/4 of the total RNA amount present in an aliquot of 44 µL axolotl extract (35–40 µg). Following a 4 hr incubation, the reaction sample was extracted by LiCl method, then analyzed by ethidium bromide staining/UV illumination and autoradiography. As shown in figure 4A (lane 3), a radioactive signal migrating at the same size as the 555 bases long heterologous RNA was clearly detected. This signal was absent when no exogenous RNA was added to the reaction (lane 2). In this case, the intensity of the signals detected as discrete bands in the axolotl extract appeared higher in absence of exogenous RNA in comparison to the intensity of the same signals detected when the exogenous RNA was present, suggesting a possible competition for the incorporation of the radioactive precursor during the RNA polymerization reaction. Cpm incorporated in TCA precipitable material measured in parallel of gel analysis are in agreement with this hypothesis since they were unchanged upon addition of large amount of exogenous RNA. Moreover, these observations were reproduced when using strongly denaturing 8 M urea-polyacrylamide gels that confirms the sizes of the radiolabelled RNAs detected (data not shown).
Similar results were obtained when 10 µg of the Xenopus globin 3′UTR transcript containing a 65 adenosines poly(A) tail at its 3′ end (620 bases) was added either to the Xenopus (Fig. 4B, lanes 4 to 7) or to the axolotl (Fig. 4C, lanes 8 to 13) extracts. The signals completely disappeared following an RNase treatment of the reaction products (Fig. 4C, lanes 11) whether they were dependent on the addition of an exogenous RNA or not. Moreover, they were sensitive to cordycepin 5′-triphosphate (i.e. 3′-deoxyadenosine 5′-triphosphate, lane 13) an RNA elongation inhibitor, but remained detectable after co-incubations of the transcript with cordycepin (i.e. 3′-deoxyadenosine, lane 12) or α-amanitin used at an RNA pol II and pol III inhibiting concentration (200 µg/mL; data not shown). This supports the involvement of cellular enzymes other than RNA pol II and III activities catalyzing the ribonucleotide triphosphate dependent step during precursor incorporation into the radiolabelled RNA polymers.
In summary, addition of large amount of exogenous RNA allows to observe a radiolabelled RNA with the same size as the added RNA. This RNA synthesis appears to occur to the detriment of the RNAs synthesized when no exogenous synthetic transcript is added.
The preceeding observations were compatible with two hypothesis : elongation of preexisting RNAs from their 3′ ends or neosynthesis of RNA. Since the elongation hypothesis depends on the presence of RNA exhibiting a free 3′OH extremity and since the RNA labelling observed could be provoked by the addition of exogenous RNA, we decided to use exogenous RNA but lacking a free 3′OH extremity. Therefore, a 3′deoxyribonucleotide (cordycepin 5′-triphosphate) was added to the 3′OH extremity of the in vitro transcript and we checked that the resulting RNA with a 3′desoxy modified end (3′H end) was unable to incorporate [α–32P] ATP in a classical in vitro polyadenylation reaction. Synthetic globin and c-myc RNA, with 3′OH or 3′H ends, were incubated in vitro in the presence of [α–32P] ATP and poly(A) polymerase and further analyzed by electrophoretic migration and autoradiography. As shown in figure 5A, the radioactive labelling detected for the unmodified 0.62 and 2.2 kb RNA (lanes 1 and 3) indicated that our poly(A) tailing conditions were efficient. Therefore the very low labelling detected for the 3′H RNA (lanes 2 and 4; less than 1% of the signal registered for lanes 1 and 3) demonstrated the efficiency of the 3′end modification of these RNAs. When we used RNAs with their 3′ends modified or not in standard RNA polymerization assays with amphibian extracts, we observed radiolabelled RNAs migrating as the exogenous RNAs, whatever the 3′ends of the exogenous RNA used during the incubations (Fig. 5B). This observation therefore supports the RNA neosynthesis hypothesis.
We then compared the intensity of the radioactive labelling detected in axolotl polymerization reactions containing different combinations of exogenous transcripts with 3′OH or 3′H ends. We reasonned that in the neosynthesis hypothesis, an equal amount of each Xenopus synthetic globin (0.62 kb) and c-myc (2.2 kb) RNA would result in identical labelling intensities of the 0.62 kb and 2.2 kb radiolabelled signals (ratio=1) whatever the 3′ ends of the transcripts. However, since an equal amount of each Xenopus synthetic RNA corresponds to about 4 fold more globin 0.62 kb transcript as compared to the c-myc 2.2 kb RNA (i.e. 4 fold more 3′ globin extremities as compared to 3′ myc extremities), the elongation hypothesis would result in a radiolabelling intensity of the 0.62 kb radioactive signal 4 fold higher than that of the 2.2 kb signal (ratio=4) if both transcripts harbored 3′OH ends. We first tested the co-incubation of 13 µg of each unmodified Xenopus RNA in the axolotl extract in the presence of the radioactive precursor (CTP). As shown in figure 5B (lane 4), the intensity of the radioactive 0.62 kb RNA signal was similar to the 2.2 kb RNA signal, with a ratio around 1.4. Identical ratios were obtained when equal amounts (13 µg) of two exogenous RNAs, one having an unmodified 3′OH end and the other one a modified 3′H end, were incubated during 3 hrs in the axolotl extract (lanes 5–6 and 7–8; ratios : 1.18 and 1.27, respectively) or in the Xenopus extract (lanes 9–10; ratio : 1.15). Therefore, these results confirm that the RNA synthesis is not due to a template independent terminal labelling at the 3′OH-end of the exogenous RNA.
To further investigate the nature of radiolabelled RNAs, we wished to know if the detected RNAs could find a complementary strand in axolotl LSE. We used an experimental approach inspired by classical RNase protection protocols to allow RNA duplex hybridization within the total RNA pool extracted following a 3 hr RNA polymerization reaction. Moreover, we chose to use a large amount of an exogenous synthetic RNA during the RNA polymerization reaction since this favors the synthesis of a radiolabelled RNA at the same size as the exogenous RNA (see Fig. 4). To overcome artefactual secundary structures stabilized during the phenol extraction step, extracted RNAs were heat-denatured and renaturation was performed overnight before RNase A treatments. The range of RNase A concentrations chosen allows to distinguish between single stranded and double stranded RNAs. In this experiment (Fig. 6), the RNA radiolabelled at the end of the incubation with axolotl LSE was expected to be totally hydrolyzed at 100 ng/mL RNase A if, as was observed for the control sense 534 bases RNA alone (lane 12), the radiolabelled RNA is single stranded at the end of the renaturation step. Conversely, the radiolabelled RNA was expected to resist at 100 ng/mL RNase A if it was able to find a complementary strand, like the two reverse complementary single stranded RNAs used as controls (lane 17). The phosphoImager scan clearly shows that the radiolabelled RNA detected at the same migration length as the exogenous RNA used during the RNA polymerization reaction is degraded at 100 ng/mL RNase A (Fig. 6, lane 7). This RNA is therefore single stranded RNA and, in particular, does not form RNA duplexes with the exogenous RNA present in large amount during the hybridization reaction. Accordingly, the long sized radiolabelled RNAs have to be mostly single stranded RNAs and not dsRNAs at the end of the RNA polymerization reaction catalyzed by amphibian extracts.
It is the first time that low speed extracts (LSE) from unfertilized eggs are performed in the urodele axolotl. To characterize the replicating properties of these extracts, classical DNA templates were incubated in polymerization reactions using axolotl LSE and parallel incubations with Xenopus LSE as controls. Axolotl LSE is unable to replicate DNA from demembranated Xenopus sperm nuclei whether Ca++ was added to the incubation mix or not. In a Xenopus LSE, incubation of demembranated sperm nuclei leads to the loading of prereplication complexes on DNA and Ca++ addition allows the Cdk (cell-cycle dependent kinase) dependent initiation of DNA replication through activation of the licensing factor , . Preliminary experiments indicated that axolotl LSE were indeed not responsive to Ca++ addition : both the levels of the phosphorylated forms of MAP kinases and Cdk1 (on tyrosine 15) that accumulate in axolotl UFE  remained unchanged in axolotl extracts following Ca++ addition (data not shown), although they are modulated when intact axolotl UFE are activated . This observation therefore raises the possibility that at least one pathway necessary to the coordinated regulation of Cdk was disrupted during axolotl LSE preparation. We did not prepare extracts from axolotl UFE activated prior to low speed extraction but this would certainly be required to further examine the DNA replicating properties of axolotl extracts.
Considering the molecular requirements for DNA dependent DNA polymerization, it was anticipated that SS M13 DNA would allow DNA polymerization driven by DNA primase and DNA polymerase activities of axolotl LSE. When 150 ng of SS M13 DNA are added to the axolotl extract, the SS M13 DNA is fully converted in DS DNA (Fig. 1B). These results show that one round of polymerization occurs on each input SS DNA and probably only one since DS DNA cannot be used as template by axolotl LSE.
Our results show for the first time that axolotl and Xenopus extracts are intrinsically able to synthesize RNA since addition of a radiolabelled RNA precursor to the extracts allow registration of TCA precipitable cpm that i) are sensitive to 5′-triphosphate cordycepin, an RNA chain elongation inhibitor and that ii) totally disappear following RNase treatment. Previous works showing RNA synthesis in amphibian extracts indeed study transcription and its regulation, i.e. DNA dependent RNA synthesis , , . In these experiments, the RNA polymerase activity that would not depend on the addition of a DNA template is intentionaly kept at a very low detection level. On the contrary, our experiments show that the incorporation rate into RNA polymers observed without addition of an exogenous template to amphibian extracts represents 1% of the input radioactivity following a 6 hr incubation as compared to 0.1% at t=0 of the kinetics. In the experiments designed to visualize the incubation products, this low incorporation rate requires long exposure times, especially to observe the long sized radiolabelled RNAs (Fig. 3 to to6).6). In these conditions, the presence of discrete bands ranging from 0.8 to 2.7 kb was detected and we were able to observe an additional band when adding a large amount of exogenous RNA to the extracts. If this indicates that high RNA concentrations are important for the RNA labelling, this is not always true since no radioactivity is detected comigrating with the 28S RNA (4 kb in axolotl) whose amount is equivalent to the 18S RNA, nor with endogenous 350/500 bases RNA whose amount is also high in the extracts (Fig. 3). The RNA synthesis we report is therefore somehow correlated to some but not all the RNAs present at high concentrations in the in vitro assay.
If Xenopus extracts are well known to promote transcriptional repression due to the large store of histones and the resulting assembly of DNA into chromatin , , pol II and III promoter repression can be released upon histone depletion , ,  or TFIIIA addition , thereby demonstrating the existence of RNA polymerase II and III activities in amphibian extracts. However, these activities do not probably account for our observations because despite the use of α-amanitin doses known to inhibit them , the RNA labelling is still observed.
The detection of radiolabelled RNA comigrating with synthetic or endogenous transcripts present at high concentrations in the amphibian extracts raised the possibility that the RNA synthesis corresponded to the template independent labelling of primer-RNAs at their 3′-ends. This could be due to various enzymatic activities : the first poly(A) polymerase activity identified in vertebrate cells  shows a relative fidelity to ATP incorporation in mRNA poly(A) tails but it is also able to incorporate CTP and such an activity exists in the nucleus and cytoplasm of frog oocytes . A polyadenylation activity using CTP and UTP for polymerization of tails at the 3′-ends of ribosomic RNA has been characterized in vertebrates . RNA specific uridylyl transferases are also template-independent polymerases that add ribonucleotides to the 3′ ends of RNA molecules . We tested the 3′ end labelling hypothesis by using large amounts of exogenous RNA harbouring 3′deoxy-ends (Fig. 5). At least for the RNA synthesis dependent on exogenous RNA addition, our results clearly show that the detection of discrete bands does not depend on the 3′OH-end elongation of the exogenous RNA added to the polymerization mix. Moreover, since the labelling of RNA comigrating with exogenous transcripts seems to occur at the expense of the labelling observed when no exogenous RNA is added to amphibian extracts (Fig. 3), we considered that only one enzymatic activity may be involved in the CTP incorporations.
Overall our results therefore suggest that the RNA synthesis does not depend neither on DNA-dependent RNA polymerization activities nor on template-independent 3′OH-end long-sized-RNA extension activities. The RNA labelling could therefore correspond to an RNA template dependent RNA synthesis.
While no homolog of the plants, fungi, S. pombe or C. elegans RNA dependent RNA polymerases (RdRp) (for a review see ) has been identified from the Drosophila to the mammalian genomes, cellular proteins harboring RdRp activity have been characterized. In the absence of RdRp encoded by the virus, the RNA templated replication of Hepatitis Delta virus RNA genome depends on cellular enzymatic activities. Several teams have documented RNA dependent RNA synthesis of the antigenomic RNA that is insensitive to α-amanitin as well as RNA dependent RNA synthesis of the subgenomic RNA that is sensitive to the drug, both of them implicating cellular RNA polymerase II associated with a viral protein , , , . Moreover, purified RNA Pol II exhibits RdRp activity whose molecular basis has been shown to reside at the active site used during transcription . Prompted by the need to explain the RNAi in Drosophila, a recent work has identified the largest of the three subunits in the RNA polymerase II core elongator complex elp-1/IKAP as an RdRp activity involved in transposon suppression . An RdRp activity was also evidenced for human telomerase reverse transcriptase catalytic subunit (TERT) towards RNA templates that are single stranded transcribed RNA with reverse autopriming properties .
In axolotl and Xenopus oocytes or UFE, we have previously provided evidence for an in vivo RNA polymerization that generates additional copies of RNA and could be part of a general RNA degradation effective during early development , . As it has been observed in vivo, our in vitro results are ascribable to an asymetric RNA synthesis process. Indeed we show here that the radiolabelled RNA with the same size as the exogenous transcript is mostly single stranded, i.e. corresponds to RNA of a single polarity. Since the RdRp activities of TERT or elp-1/IKAP produce dsRNA, this cannot explain the RNA polymerization catalyzed in the amphibian LSE. Nevertheless, RNA dependent RNA synthesis is asymetric in viral RNA replication  and for RNA amplification of cytoplasmic β-globin RNA in murine erythroleukemia cells , . The molecular steps necessary to explain the observations that we present here in amphibian remain to be elucidated. However, since 3′OH-end modification of the added RNA does not preclude the observation of RNA synthesis, none of these steps is strickly dependent on the 3′OH-end of long sized RNAs. In particular, priming of RNA synthesis involving an intramolecular foldback RNA hybridization as a first step can be already excluded.
A difference exists in the effectiveness of the RNA dependent RNA synthesis between the observations we made in amphibian in vitro and in vivo systems. While it is possible to register in vivo a quantitative amplification of the injected RNA, this observation is not reproduced in vitro : the intensity of the Stains-all band corresponding to the exogenous transcript is not higher at the t=3 hr point of the incubation than at t=0 (Fig. 6). This result suggests that at least one step necessary to RNA synthesis is a limiting step in vitro. One protein may have lost its activity during the extraction or could be inhibited by a co-extracted component of the extracts. Optimization of RNA synthesis conditions in vitro will be useful to understand the mechanisms that command such RNA polymerization in vertebrates.
Xenopus extracts incorporate [α−32P] CMP into TCA precipitable material. A. Time course of incorporation of [α−32P] CMP in two Xenopus LSEs. Aliquots (5 µL) of a standard reaction (50 µL) using [α−32P] CTP were withdrawn throughout kinetics ranging from t=0 to t=180 minutes (min), processed for [α−32P] CMP incorporation into acid-insoluble material by TCA precipitation and the [α−32P] CMP incorporated was expressed as counts per minute (cpm) for 106 cpm used in the reaction (106 cpmtotal). B. Incorporation of [α−32P] CMP in four independent experiments using the same Xenopus mitotic LSE. Results obtained using the same LSE during 4 hr incubation independent experiments (1 to 4) are shown as histograms (white bars) where [α−32P] CMP incorporated into TCA precipitable material was expressed as counts per minute (cpm) for 106 cpm used in the reaction (106 cpmtotal). Hatched bars correspond to samples incubated without exogenous RNA and processed in parallel of the standard incubation 2. White bars surrounded by a bold line (m) represent the mean of the different values with the corresponding standard deviation obtained at t=0 h and t=4 h in incubations with exogenous RNA. C. [α−32P] CMP is incorporated into RNA. Using a Xenopus LSE in a reaction (50 µL) with [α−32P] CTP, aliquots (10 µL) were collected at t=0 and t=4 h. [α−32P] CMP incorporated into TCA precipitable material (cpmTCA) was determined using a standard protocol (white bars) or a modified one including a further incubation at 37°C with RNase (black bar) or with RQ1 DNase (grey bar) prior to TCA precipitation. Similar results were obtained using another Xenopus LSE.
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The authors are grateful to L. Dandolo and G. Hervé for helpful comments and for critical reading of the manuscript. They thank M. Méchali for welcoming YA in his laboratory to learn LSE technologies, S. Bocquet for technical help to prepare Xenopus LSE extracts, S. Caulet for his competence in using various softwares, E. Manzoni and S. Authier for their competence in maintaining the axolotl colony.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by a grant from the Association pour la Recherche sur le Cancer (ARC 3387). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.