Reverse protection assay: a tool to analyze transcriptional rates from individual promoters
© Zubo et al; licensee BioMed Central Ltd. 2011
Received: 10 October 2011
Accepted: 20 December 2011
Published: 20 December 2011
Transcriptional activity of entire genes in chloroplasts is usually assayed by run-on analyses. To determine not only the overall intensity of transcription of a gene, but also the rate of transcription from a particular promoter, we created the Reverse RNase Protection Assay (RePro): in-organello run-on transcription coupled to RNase protection to define distinct transcript ends during transcription. We demonstrate successful application of RePro in plastid promoter analysis and transcript 3' end processing.
Today's molecular biology employs several methods to determine the steady-state levels of RNAs, such as Northern- and dot-blot analysis, ribonuclease protection assay (RPA), reverse transcription-PCR (RT-PCR), and quantitative real-time PCR. These methods differ in their sensitivity and information content. For example, while RT-PCR allows quantification of RNA molecules of different lengths, RPA permits determination of the relative amount of transcripts with distinct 5'- and/or 3'-ends . In general, RPA is based on hybridization of a labeled, single-stranded antisense RNA probe to the target RNA. Subsequent incubation with an RNases mix degrades those RNA molecules that do not form double-stranded hybrids. Final inactivation of RNases and precipitation of the protected RNA hybrids is followed by electrophoretic analyses, which reveals the presence, size, and relative level of RNA that was protected by the antisense probe . The RPA method has been modified to serve for different tasks, such as measuring the radioactive signals by scintillation counting [RiPPA method; ], and replacing radiolabeled with biotinylated probes . Adding constitutively transcribed or in vitro synthesized RNA as an internal standard during RNA isolation allows quantification of the RNA analyzed by RPA [4, 5].
However, the overall RNA content reflects a balance between the synthesis and degradation of transcripts . The aforementioned methods only identify RNA steady-state levels. To directly evaluate the rate of individual gene transcription, run-on transcription is utilized: transcripts are labeled by adding radio-nucleotides during a brief time of incubation and subsequently analyzed by dot-blot hybridization . This technique applies to all genetic compartments such as bacterial cells, nuclei, mitochondria, and chloroplasts. However, this method also has some limitations. The double-stranded probes usually used for dot-blots in run-on assays are able to hybridize with both sense and antisense transcripts. Interfering transcription initiated by adjacent promoters on the opposite strand may disturb the results obtained by this method. This is especially significant for transcription of bacterial and chloroplast genes, where a great amount of antisense transcripts are generated [e.g., [7, 8]]. Furthermore, the size of transcripts analyzed and the corresponding promoter(s) remain unknown.
Generally, plastids possess a rather inefficient transcriptional termination system. Although inverted repeat sequences, which can fold into stem-loop structures similar to bacterial terminators, are often found at 3' ends of plastid transcripts, they do not serve as terminators, but rather function as RNA 3' end processing and maturation signals [9–11]. Leaky termination might therefore be one of the reasons for the observed dual transcription of certain genes both from their own promoter and as part of a polycistronic operon [7, 12–19].
We have previously shown that synthetic cytokinin 6-benzyladenine (BA) enhanced transcription rates of several chloroplast genes in barley. Among the most affected was the rrn16 operon . To date, the only promoter known to drive transcription of the rrn16 operon in barley is a sole PEP promoter [Prrn16-118/-119; , PEP, plastid-encoded plastid RNA polymerase; reviewed in [22, 23]]. However, directly upstream of the rrn16 operon, the gene for tRNAVal (GAC) is located (Hordeum vulgare plastid genome: GenBank EF115541). To investigate if the target of cytokinin action to activate rrn16 gene expression is increased transcription initiation from Prrn16-118/-119, we developed a novel method, which couples transcription and RNA mapping, the Re verse RNA Pro tection assay (RePro). Essentially, RePro is a combined technique of in-organello run-on transcription in the presence of radiolabeled nucleotides and an R Nase P rotection A ssay (RPA) using unlabeled RNA probes, with the advantages of both of these methods. In principle, RePro allows the accurate analysis of the rate of transcription from individual promoters, which is a crucial step in studies of regulation of gene expression.
Results and discussion
Mapping of 5'-ends of rrn16, rbcL, and psbA transcipts by RePro
Origin of non-specific background signals in the RePro
To investigate the influence of the integrity of the chloroplasts, RePro with chloroplasts isolated from the Percoll gradient as intact or broken were carried out (Figure 3A). Since the broken chloroplasts displayed a much lower transcriptional activity (Figure 3A, lane 6), only intact organelles should be used as a source for run-on transcription (Figure 3A, lane 3). However, the run-on transcription reaction is performed in a chloroplast lysate, because of a sharp drop in the osmotic strength when the isolation buffer is replaced by transcription buffer. To test if the chloroplast integrity is further required, we performed RePro assays where sorbitol was added to transcription buffer to the final concentration of 0.33 M to avoid osmotic lysis. Evidently, lysis of the chloroplasts before or during in vitro transcription is necessary to allow fast and easy accessibility of exogenously added (radiolabeled) nucleotides to the plastid transcription system [lanes 4 and 5; ].
Optimization of the unlabeled asRNA amounts in RePro
The amount of labeled asRNA probes in conventional RPAs does usually not exceed 1 ng . To determine the best amount of unlabeled asRNA, RePro assays with increasing amounts of unlabeled asRNA were performed (Figure 3B). An increase from 0.01 to 0.1 μg of the unlabeled asRNA enhanced the signal by approximately 30% (Figure 3B, lanes 3, 4). A further increase in the asRNA amount to 1 μg did not result in further signal enhancement (Figure 3B, lane 5), which indicates saturation of the system. The asRNA should never be the limiting factor for hybridization with the corresponding chloroplast transcripts labeled by run-on transcription. Otherwise, it would not be possible to compare distinct transcriptional rates. Therefore, we typically added 0.1 μg of unlabeled asRNA to [32P]-UTP-labeled chloroplast RNA isolated from run-on transcription with about 5 × 108 chloroplasts.
RePro exhibits similar increases in signal over time as run-on analysis
Unlike the run-on assay, RePro does not fully reflect the accumulation of full-length and/or elongated but processed transcripts, but of relatively short 5'-fragments defined by the design of the asRNA for the protection assay. In our experiments, a 184-nt fragment of the rrn16 transcript 5'-end was protected. Therefore, the effect of transcript elongation on the detectable amount of freshly transcribed RNAs may be limited. Moreover, it is generally believed that incorporation of [32P]-label in run-on assays only occurs during elongation of already initiated transcripts [29, 30]. RNA synthesis is greatly reduced after chloroplast isolation (performed at 4°C) and is restored under temperature conditions of about 25°C. Supposedly, the rate of the reaction during the first 10 min reflects the intensity of RNA transcription. Thereafter, RNA degradation and/or accumulation start to influence the observed amounts of labeled transcripts . Transcription due to new initiation by plastid RNA polymerases during the run-on process occurs slower than in planta is therefore considered insignificant .
Decline of transcriptional activity in isolated chloroplasts
To evaluate the robustness of the plastid in vitro transcription system over a time course and to compare the analytical methods of run-on and RePro, we pulsed in vitro transcription assays by successively adding [32P]-UTP during a 50 min time course (Figure 4B). To measure the rate of transcription during first 10 min, we added [32P]-UTP at the moment of the reaction start. To measure the rate of transcription during subsequent 10-min intervals, we firstly performed the transcription reaction for 10, 20, 30, or 40 minutes with unlabeled nucleotides, followed by an additional incubation of 10 min with added [32P]-UTP. RNA isolated from each sample was processed as described for run-on (black graph) and RePro analyses (grey graph). Both methods detected a comparable, nearly linear decline of about 80% of the transcriptional activity in isolated chloroplasts during the 50 min of incubation. Interestingly, a less pronounced slope of the transcriptional activity was observed after 30 min (20 min without label, followed by 10 min with [32P]-UTP), a point of time which showed increased transcript accumulation when incubated the entire period of 30 min with [32P]-UTP (Figure 4A). This suggests that stabilization of rrn16 RNAs generated by run-on transcription significantly occurs after 20 to 30 min of reaction time. However, during further incubation up to 60 min, the RNA stability seemed to decline again. Since only label incorporated into the protected RNA 5'-ends of 184 nt contributes to the RePro signal, it might be possible that initiation of new transcripts is slower than the elongation of transcripts initiated in planta, which are still detectable by run-on but not with RePro.
Pulse-chase experiments to determine the stability of synthesized RNAs
The surprising effect of still rising amounts of labeled rrn16 transcripts 15 min after adding the unlabeled UTP (Figure 5B and 5D), which was not observed using the conventional run-on technique, might partly be explained by a transcriptional arrest caused by UTP deficiency in the in vitro transcription system. Adding unlabeled UTP might remove the elongation arrest and RNA synthesis resumes. These partially labeled transcripts are now fully detectable by the antisense RNA probe and may therefore more substantially add to the weak reverse protection signal as compared to the solid phase hybridization signal in standard run-on analyses. Indeed, an additional signal of about 130 bp appears in samples without the addition of unlabeled UTP, but is absent from samples where unlabeled UTP was added (Figure 5B, asterisk). In run-on assays, however, such an increase of the rrn16 transcript signal was not observed 15 min after adding unlabeled UTP (Figure 5A and 5C). Two reasons may account for this: firstly, the length of transcripts is unimportant for hybridization to the probe on the dot blot; secondly, the amount of labeled transcripts then being able to hybridize after lifting the arrest is too small to contribute significantly to the entire signal.
RePro assays are capable of both determining transcription rates of individual promoters and monitoring RNA processing
We developed a novel method, the Re verse RNA Pro tection assay (RePro), which allows the accurate analysis of distinct transcript 5'- and 3'-ends during the transcriptional process by combining run-on and RNase protection assays. Using RePro, we were able to show that about 10% of the transcriptional activation of the rrn16 gene by cytokinin comes from read-through transcription initiated upstream of its Prrn-118/-119 promoter. Furthermore, although the important initial step of RePro, the run-on assay, is thought to be a system of transcription elongation, we clearly observed substantial contribution of in vitro transcription initiation. We additionally demonstrated that RNA degradation processes did not affect the results obtained by RePro.
In general, application of RePro allows an additional analysis of gene transcription, i.e., testing transcription from individual already known promoters and processing of freshly generated transcripts. The effects of transcription from other promoters of the gene and nonspecifically transcribed sense or antisense RNA synthesis are completely excluded. RePro reflects changes occurring at the level of in vitro transcription initiation more adequately.
Plant growing and benzyladenine treatment
Experiments were performed with primary leaves of 6- to 9-day-old barley plants (Hordeum vulgare L., cv. Luch), which were grown in soil at 20°C under illumination of 270 μmol m-2 s-1 from luminescent tubes (Lamp Master HPI-T Plus 400 W E40, Philips) under a 16-h photoperiod. For experiments on transcriptional regulation by cytokinin, primary leaves were detached from 9-day-old plants and incubated on filter paper moistened with water under continuous illumination of 270 μmol m-2 s-1 for 24 h. Subsequently, leaves were transferred to water or 2.2 × 10-5 M BA and kept for 3 h under the same light conditions.
Run-on - chloroplast isolation
Apical segments (2 cm in length) of the first leaves detached from 9-day-old barley plants were used for chloroplast isolation, because preliminary experiments have demonstrated that this leaf part is most sensitive to cytokinin treatment . Leaf segments (10 g) were homogenized in 80 ml of buffer A (0.33 M sorbitol, 50 mM Tricine, pH 8.0, 2 mM EDTA, and 5 mM β-mercaptoethanol). The homogenate was squeezed through Miracloth (Calbiochem, San Diego, CA, USA) and centrifuged at 2700 g for 6 min. The pellet was resuspended in 1.5 ml of buffer A and fractionated in a 40/70% discontinuous Percoll gradient by centrifugation at 4000 g for 30 min. Intact chloroplasts were collected at the interface between 40 and 70% Percoll, washed in buffer A, and resuspended in 0.5 to 1 ml of buffer B (50 mM Tris-HCl, pH 7.0, 10 mM MgCl2, 10 mM KCl, and 4 mM β-mercaptoethanol). The number of chloroplasts in the samples was determined by counting the organelles under a light microscope using a Fuchs-Rosenthal hemocytometer . Chloroplasts (5 × 108) were precipitated by centrifugation, resuspended in 45 μl of buffer D (50 mM Tris-HCI, pH 7.0, 10 mM MgCl2, 10 mM KCl, 4 mM β-mercaptoethanol), and used for run-on transcription. All procedures were performed at 4°C.
Run-on - in vitro transcription reaction
In vitro transcription reaction with 5 × 108 lysed plastids was carried out in a 100 μl volume by the method of Mullet and Klein  modified according to Zubo et al. . Transcription was performed for 10 min at 25°C in buffer containing 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 0.2 mM CTP, GTP, ATP, 0.01 mM UTP, 50 μCi of [α-32P]-UTP (≈3000 Ci/mmol, Amersham, UK), 20 U of RNasin (Fermentas, Lithuania), and 10 mM β-mercaptoethanol. The reaction was stopped by the addition of an equal volume of stop buffer (50 mM Tris-HCl, pH 8.0, 25 mM EDTA, and 5% sarcosyl). [32P]-labeled transcripts were isolated from chloroplasts as described by Guadino and Pikaard . Reaction products were extracted twice with phenol/chloroform and once with chloroform. RNA was precipitated by an equal volume of isopropanol in the presence of 0.1 volume of 3 M sodium acetate, pH 6.0, and 20 μg of tRNA.
Blotting of plastid gene probes, their positions, and hybridization conditions in conventional run-on experiments were carried out as described in Zubo et al. . Radioactive signals of dot blot assays were detected and quantified using a Molecular Imager FX and the Quantity One software (Bio-Rad, USA). The results, termed 'relative transcription activity', of three independent biological with two technical replicates were analyzed and visualized using Microsoft Excel (see Figures 4, 5, and 6).
RNAse protection reaction
The assay was performed according Sambrook et al. , however, with slight changes. Performed as control reactions, RNase protection assays contained 1 μg (rrn16) or 5 μg (psbA and rbcL) of chloroplast RNA and [32P]-UTP-labeled of the respective asRNA (about 3 × 105 cpm, MEGAscript T7 kit, Ambion), which were subsequently co-precipitated with ethanol, dissolved in 30 μl hybridization buffer (40 mM PIPES, pH 6.8, 1 mM EDTA, pH 8.0, 0.4 M NaCI, 80% deionized formamide), denatured (5 min 90°C), and incubated at 60°C over night. After adding 300 μl RNase digestion buffer (300 mM NaCl, 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, pH 7.5) and 5 μl of a RNaseA/RNaseT1 Mix (Fermentas, Lithuania; 2 mg/ml of RNase A, 5000 U/ml of RNase T1), digestion of single-strand RNAs was performed by incubation at 30°C for 1 hour. The digestion was stopped by adding 20 μl of 10% SDS and 10 μl of 10 mg/ml proteinase K (Fermentas, Lithuania) and further incubation at 37°C for 30 min. The RNAs were treated with phenol/chloroform, ethanol precipitated in the presence of 20 μg of carrier yeast tRNA (Invitrogen), and resuspended in 10 μl of gel-loading buffer (95% deionized formamide, 0.025% bromophenol blue, 0.025% xylene cyanol, 5 mM EDTA, pH 8.0, 0.025% SDS). Protected RNA fragments were fractionated in 4% polyacrylamide/8 M urea gels in Tris-borate buffer, pH 7.7. After exposure, the gels were analyzed with a PhosphoImaging system and specific bands and signals quantified using the complementary software (Quantity One, BioRad). The results of three independent biological with two technical replicates were analyzed and visualized using Microsoft Excel.
In case of the reverse RNase protection assay, the [32P]-UTP-labeled RNA synthesized in a run-on experiment in vitro and unlabeled asRNA (from 0.01 to 1 μg) were co-precipitated at -20°C for 1 h and subsequently handled as outlined for the RNase protection assay.
The length of protected RNA fragments was determined using the RNA Century™ Marker Templates (Ambion, USA) according to the manufacturer's protocol.
In vitro antisense RNA synthesis
Synthesis of antisense RNA (asRNA) was performed using the MEGAscript T7 Kit (Ambion, USA) according to the manufacturer's protocol using PCR fragments comprising the promoter region of the gene studied as templates. Each reverse primer contained the sequence of the T7 promoter (lowercase letters). The following primers were used (for positions see also Figures 2 and 7):
rrn16-for CGAGCGAACGAGAATGGATAAGAG, rrn16-rev cagagatgcataatacgactcactatag ggagaCGACTTGCATGTGTTA; rrn16-e-up GTAATCGCCGGTCAGCCATAC, rrn16-e-low cagagatgcataatacgactcactatagggagaTGAAGAAGTGTCAAACC; psbA-for CCGACTAGTTCCGGGTTCGAG, psbA-rev cagagatgcataatacgactcactatag ggagaTTGTACTTTCGCGTC; rbcL-for TAATTTGGGTTGCGCTATACCTATCA, rbcL-rev cagagatgcataatacgactcactatag ggagaTTGAGGGCATGCT. After synthesis and treatment with DNase I, the [32P]-UTP-labeled asRNA was purified by electrophoresis in a denaturing polyacrylamide gel to remove shorter than full-length RNAs as well as the DNA template. RNA was eluted from the gel by gel fragment incubation in buffer containing 1 mM EDTA and 0.2% SDS (350 μl per gel fragment) overnight at 37°C with constant weak shaking. In the case of unlabeled asRNA, the procedure of electrophoretic purification was omitted. Firstly, no additional bands are seen after electrophoresis if the DNA template protects unlabeled asRNA, since the chloroplast RNA is labeled but not the asRNA. Secondly, unlabeled RNA is more stable because of the absence of its self-degradation by radioactive radiation.
This work was funded by Deutsche Forschungsgemeinschaft (SFB 429 to KL and TB). We thank Christian Schmitz-Linneweber for helpful discussions.
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