Pelczar H et al. (2010), Evidence for an RNA polymerization activity in ...

XB-ART-42569

PLoS One 2010 Dec 17;512:e14411. doi: 10.1371/journal.pone.0014411.

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Evidence for an RNA polymerization activity in axolotl and Xenopus egg extracts.

Pelczar H , Woisard A , Lemaître JM , Chachou M , Andéol Y .

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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).

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Species referenced: Xenopus
Genes referenced: matn1 myc

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	Figure 1. Axolotl low speed extracts (LSE) polymerize DNA when single-stranded M13 DNA is added to the extracts.A : Cpm incorporated into TCA precipitable material were registered following 2 hr incubation reactions (44 µL) performed at 20°C in the presence (+) or not (−) of the mentionned DNA template, [α–32P] dCTP as DNA precursor, as well as CaCl2 addition (+) or not (−). LSE prepared from Xenopus UFE and incubated with demembranated sperm nuclei provided the control for dCMP incorporation conditions as well as for activation of LSE by CaCl2 addition. Each histogram corresponded to 2 (Xenopus) to 5 (axolotl) experiments. B : Single-stranded (SS) M13 DNA (150 ng) were mixed in a reaction (88 µL) with axolotl LSE. The reaction was divided into equal aliquots incubated at 20°C during 2 hr (2h, lanes 6) or not (t = 0, lanes 5). A reaction (44 µL, t = 0) without any M13 DNA addition to the extract was also processed (lanes 4). Following phenol/chloroform extractions and ethanol precipitation, the DNA content of each aliquot was analyzed by gel electrophoresis in an 1% agarose gel. Circular M13 DNA either single-stranded (SS; lanes 1 : 100 ng) or double-stranded (DS; lanes 2 : 300 ng) or linearized EcoRI-digested DS M13 (lanes 3 : 400 ng) were electrophorezed in parallel as well as 1 kbp ladder molecular marker (Fermentas, M). Left side : photograph under UV illumination after ethidium bromide staining of the gel. Right side : autoradiograph following gel transfer and hybridization of the corresponding membrane to a radiolabelled SS M13 DNA probe. The migration lengths corresponding to the different forms of M13 DNA are indicated with black diamonds (SS M13), asterisks (supercoiled form of DS M13, I), black squares (closed or nicked relaxed forms of DS M13, II) and black arrows (linearized DS M13, III).
	Figure 2. Axolotl extracts incorporate [α–32P] CMP into TCA precipitable material.A. Time course of incorporation of [α–32P] CMP in different axolotl LSEs. Aliquots (5 µL) of a standard reaction (50 µL) using [α–32P] CTP were withdrawn throughout kinetics ranging from t = 0 to 2 hours (h), 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). Curves with a thin line corresponded to the kinetics obtained with individual extracts and the mean curve is represented in bold with a standard deviation. B. Incorporation of [α–32P] CMP in seven independent experiments using the same axolotl LSE. Results obtained using the same LSE during 4 hr-incubation independent experiments (1 to 7) are shown as histograms (white bars) where [α–32P] CMP incorporated into TCA precipitable material is 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 incubations 1, 2 and 5. White bars surrounded by a bold line (m) represent the mean of the different values, with the corresponding standard deviation, obtained at t = 0 and t = 4 h in incubations with exogenous RNA. C. [α–32P] CMP is incorporated into RNA. Using an axolotl LSE in a reaction with [α–32P] CTP (50 µL), 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 water (grey bar) prior to TCA precipitation. Similar results were obtained using two other axolotl LSEs.
	Figure 3. Discrete radiolabelled RNA bands are detected after incubation of the axolotl and Xenopus extracts in the presence of [α–32P] CTP or [α–32P] UTP.Polymerization reactions were performed using axolotl or Xenopus LSE with either [α–32P] CTP or [α–32P] UTP (lane 4) and in the presence (lane 6) or not of 200 µg/mL α-amanitine, an RNA pol II and pol III inhibitor. After the indicated incubation time, samples were treated at 37°C during 1 hr with RNase A (lane 5) or during 15 min with RQ1 DNase (lane 8) or not (lanes 1–4, 6, 7, 9–11). RNA was extracted, electrophorezed through a denaturating agarose gel and visualized after ethidium bromide staining and UV illumination; the gel was dried and autoradiographied. Migration lengths of RNA size markers are indicated in bases (b).
	Figure 4. A radiolabelled RNA comigrates with the exogenous RNA added to the extract.Reactions were performed during 4 hr with axolotl (A : lanes 1–3. C : lanes 8–13) or Xenopus (B : lanes 4–7) LSE in the presence of [α–32P] CTP, in the presence (+) or absence (−) of 10 µg of in vitro transcribed Xenopus globin 3′UTR poly (A)− (555 b; lanes 1–3 and 8–13) or poly (A)+ (620 b; lanes 4–7) RNA and in the presence of 2.5 mM cordycepin (lane 12) or cordycepin 5′-triphosphate (lane 13) or not (lanes 1–11). After incubation, samples were treated at 37°C during 1 hr with 10 mg/mL RNase A (lane 10) or not, RNA was extracted and processed according to the same protocol as in figure 3. The radioactivity corresponding to 1/10 of the reaction analyzed by TCA precipitation is indicated in A (counts per minute : cpm). In vitro synthesized Xenopus globin 3′UTR poly(A)− (555 b) or poly(A)+ (620 b) RNA analyzed in parallel are shown in the UV part of A and C respectively. Migration lengths of RNA size markers (M lanes in C) are indicated in bases (b).
	Figure 5. The RNA labelling does not depend on the 3′OH end of the exogenous RNA.A. Validation of the modification of the synthetic globin and myc transcripts at their 3′ends. In vitro Xenopus globin poly(A)+ (lane 1 : 750 ng and lane 2 : 1.2 µg) or myc (Myc, lane 3 : 500 ng and lane 4 : 1 µg) RNA either modified at their 3′OH end by addition of 3′deoxyATP (Glob 3′H, lane 2 or Myc 3′H, lane 4) or unmodified (Glob 3′OH, lane 1 or Myc 3′OH, lane 3) were used in a polyadenylation reaction containing [α–32P] ATP, run in an agarose gel stained with ethidium bromide and UV illuminated then immediately dried for autoradiography. The numbers (arbitrary units) indicate that less than 0.5% of the 3′ modified molecules incorporate the radioactive precursor as compared to the corresponding 3′ unmodified synthetic RNA. M = RNA molecular Markers High Range ladder. kb : kilobase. B. RNA polymerization in amphibian extracts using co-incubation of synthetic globin and myc transcripts with modified or unmodified 3′ ends. Reactions were performed at 20°C during 3 hr (h) with axolotl or Xenopus LSE (as indicated), [α–32P] CTP and different combinations of exogenous globin and myc Xenopus transcripts with 3′OH or 3′H ends (13 µg each, lanes 3–10) except when no exogenous RNA was added (lanes 1–2). After incubation, RNA was processed and analyzed as previously described (Fig. 3 and 4). The ratio (R) of the radioactive signal intensities (Glob/Myc) is indicated for each combination of synthetic RNA. Synthetic transcripts are schematized as in part A. kb : kilobase.
	Figure 6. Analysis of the radiolabelled RNA sensitivity to RNase A.RNA polymerization reactions were performed with axolotl LSE and [α–32P] CTP in the presence of exogenous sense 534 bases PKCζ synthetic transcript (A); at the indicated incubation times, total RNA was extracted, solubilized in water then either directly mixed with the loading buffer (lanes 1, 2) or diluted in the hybridizing buffer (80% formamide, lanes 3–18). Sense 534 bases PKCζ synthetic transcript was incubated in control hybridization reactions (B), either alone (lanes 9–13) or in the presence of a 540 bases synthetic RNA (antisense) complementary to the sense 534 bases transcript (lanes 14–18). RNAs were heat-denaturated then incubated overnight at 65°C. Following the denaturation/hybridization steps, the ability of the RNAs to resist to RNase A catalyzed hydrolysis was studied in the digestion buffer (8% formamide) and RNase A at final concentrations of 1 ng/mL (lanes 5, 10, 15), 10 ng/mL (lanes 6, 11, 16), 100 ng/mL (lanes 7, 12, 17), 1 µg/mL (lanes 8, 13, 18) or in the absence of RNase A (lanes 3, 4, 9, 14). RNAs were analyzed in polyacrylamide 8 M-urea gels that were stained with ≪Stains-all≫ and exposed to a PhosphoImager screen when mentioned. As compared to the RNase A ≪-≫ conditions (lane 4), the percent of residual radioactivity observed at the migration length of the sense 534 bases transcript (arrow head) has been quantitated : lane 5, 99%; lane 6, 40%; lane 7, 3%; lane 8, 1%. The sizes of the RNA molecular Markers Low Range ladder are indicated in bases (b).

References [+] :

Almouzni, Competition between transcription complex assembly and chromatin assembly on replicating DNA. 1990, Pubmed, Xenbase