- Email: [email protected]
In vitro coupled transcription splicing
Methods 37 (2005) 314–322 www.elsevier.com/locate/ymeth
In vitro coupled transcription splicing Barbara J. Natalizio a, Mariano A. Garcia-Blanco a,b,¤ a
Departments of Molecular Genetics and Microbiology, Center for RNA Biology, Duke University Medical Center, Durham, NC 27710, USA b Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA Accepted 21 July 2005
Abstract Many convincing studies published in recent years strongly support coupling of transcription and pre-mRNA processing. Despite key advances in our understanding of these processes, there is a lack of a robust in vitro system in which to study the mechanism of this coupling for complex pre-mRNAs. Here, we describe an in vitro system capable of transcribing and splicing complex transcripts with three and four exons. We also demonstrate how the system can be used to study exon silencing in vitro. We believe that this system will be a useful tool to study the mechanisms that mediate the coupling of transcription and pre-mRNA processing. 2005 Elsevier Inc. All rights reserved. Keywords: Coupling; Cotranscriptional splicing; mRNA processing; RNA polymerase II; T7 RNA polymerase; Transcription
1. Introduction The occurrence of cotranscriptional mRNA processing is widely accepted as the cytological, biochemical, and functional evidence supporting it continues to expand. The Wrst demonstration of cotranscriptional splicing came from the direct visualization of Miller chromatin spreads from Drosophila melanogaster embryos [1]. Further evidence in support of cotranscriptional splicing resulted from examining the splicing rate of transcripts from the Balbani ring 1 gene in Chironomus tentans and from the human dystrophin gene [2,3]. These studies demonstrated that while 5⬘ introns are mostly removed cotranscriptionally, the 3⬘ introns of these long transcripts are generally removed posttranscriptionally. Cotranscriptional pre-mRNA processing depends on speciWc components of RNA polymerase II (RNAP II) [4] and requires the carboxy-terminal domain (CTD) of the largest RNAP II subunit [5]. Studies focusing on the role of the CTD in processing emerged shortly after Greenleaf [6] proposed that the highly charged CTD may *
Corresponding author. Fax: +1 919 613 8646. E-mail address: [email protected] (M.A. Garcia-Blanco).
1046-2023/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2005.07.013
serve as a scaVold for pre-mRNA processing factors (reviewed in [7]). Truncation of the CTD speciWcally inhibits capping, splicing of some introns, cleavage/polyadenylation, and transcription termination (reviewed in [8–12]). Currently, most biochemical studies of pre-mRNA processing are carried out independent of transcription. Processing precursors are synthesized by bacteriophage RNA polymerases (e.g., T7 RNAP), puriWed, and subsequently incubated with cellular extracts [13–17]. The hope is that establishing a system whereby transcription and premRNA processing are functionally and perhaps, physically coupled will help elucidate connections between transcription and splicing that are currently diYcult to study by conventional in vitro pre-mRNA processing systems. To this end, we and others have developed in vitro systems to study coupling of transcription with 3⬘ end formation and polyadenylation [18,19] or with splicing of single intron premRNAs [20]. Although the latter could eYciently transcribe and splice single intron transcripts, it did not eVectively handle more complex splicing precursors that are most likely impacted by cotranscriptional coupling. With this in mind, we have developed a system that can splice larger and more complex transcripts, including three and
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four exon constructs. Our system can properly and eYciently transcribe, and splice both constitutively spliced transcripts as well as alternatively spliced transcripts. Furthermore, this system can recapitulate exon deWnition. This assay has also been used to examine trans-splicing and we observe that the delay in synthesis of the 3⬘ splice site facilitates the trans-splicing of the upstream 5⬘ splice site, as has been observed in vivo [21]. In addition, using time course analysis we can examine the order of intron removal. Moreover, we have data that are consistent with increased stability of RNAP II transcripts versus almost identical T7 RNAP transcripts. This phenomenon has been carefully documented by Hertel and colleagues also using an in vitro system (personal communication Hicks, Yang, and Hertel). This protection of the RNAP II transcripts from nuclear degradation may be attributed to the coupling of transcription and splicing. We believe that the system described here will serve as a valuable resource for studying mRNA processing in the context of coupling.
plates cleaved immediately downstream of the 3⬘UTR. Complete digestion and purity of the DNA template are imperative to the success of the downstream applications. Following digestion, templates are phenol/chloroform/isoamyl alcohol (25:24:1) extracted once, chloroform extracted twice, and ethanol precipitated with 1:10 vol of 3 M sodium acetate, pH 7.0. They are then pelleted at 14,000 rpms, washed once with chilled 80% ethanol, dried, and resuspended in nuclease-free water (Ambion) to a Wnal concentration of 1.0 g/L. Please note that we do not typically gel purify the DNA fragments of interest. Although PCR templates can be used in lieu of restriction fragments, in our experience these PCR templates do not perform as well as linearized plasmids. It is important to note that any plasmid that is normally used to express a splicing precursor in cells can be used for these in vitro reactions.
2. Description of method
Aqueous buVers and solutions are all made with nuclease-free water and are not DEPC treated.
2.2. BuVers and solutions
2.1. Plasmids/vectors DNA templates for the in vitro transcription-splicing reactions were constructed by inserting the complete human -globin gene, including both the 5⬘ and 3⬘ untranslated regions (UTRs) without the endogenous globin promoter, into pBC12/CMV [22,23] (Fig. 1). An identical -globin construct driven by T7 RNAP was created by removing the CMV promoter region and replacing it with the T7 promoter region. Additionally, a 15 nucleotide sequence devoid of pyrimidines was added to both constructs at the transcription initiation start site to prevent the polymerase from prematurely terminating. In vitro transcription-splicing reactions have also been performed with templates cloned into pcDNA3 where both the CMV promoter and T7 promoter are present in one vector and these reactions seem to work equally as well (data not shown). The -globin templates were digested both upstream and downstream of the region of interest with BsrB1 restriction endonuclease to prevent transcription from upstream cryptic promoters. This digestion cleaves approximately 400 nucleotides downstream of the 3⬘UTR of -globin. We have observed that linearizing the DNA template at this distance downstream of the 3⬘UTR results in increased splicing eYciency compared to tem-
NTP mix: 25 mM ATP and 15 mM CTP, GTP, UTP high purity nucleotides for RNA synthesis (Amersham Biosciences) (store at ¡20 °C). Mix A: 5 mM ATP and 3 mM CTP, GTP, UTP; 0.05 g/ L poly(I):poly(C) (Amersham Biosciences); 0.06 g/L poly(dI):poly(dC) (Amersham Biosciences); 50 mM creatine phosphate (Sigma Cat# P7936); 0.5 mM DTT (store at ¡20 °C). MgCl2//ZnCl2 mix: 50 mM MgCl2 · 6H2O (EMD); 1.25 mM ZnCl2 (CalBiochem) (store at ¡20 °C). Stop buVer: 100 mM Tris–HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 300 mM NaOAc, 0.75% SDS, 0.025 mg/ mL yeast tRNA (Sigma Cat# R5636) (store at 25 °C). High salt buVer: 10 mM Tris–HCl (pH 7.5), 50 mM EDTA, 100 mM LiCl, 0.5% SDS, 7.0 M urea (store at 25 °C), 7.5 M NH4 OAc (store at 25 °C). 5£ RPA hybridization buVer: 200 mM Pipes (pH 6.4), 2.0 M NaCl, 5.0 mM EDTA (store at ¡20 °C). Hybridization buVer is diluted with formamide. RPA digestion buVer: 300 mM NaCl, 10 mM Tris–HCl (pH 7.5), 5.0 mM EDTA (store at ¡20 °C). Formamide buVer: 95% formamide, 18 mM EDTA, 0.025% SDS, xylene cyanol, and bromophenol blue (store at ¡20 °C).
Fig. 1. Schematic representation of human -globin construct. Human -globin is transcribed by either RNAP II or T7 polymerase using the CMV-IE or T7 promoter, respectively. The shaded boxes represent non--globin sequence. A pyrimidine free region is inserted at the transcription start site. The last 422 nucleotides of the construct are pBC12 vector sequence. The position of the poly A site is indicated by the black box within exon 3.
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Please note that the NTP mix and mix A should not be kept for longer than one month. Furthermore, stop buVer, high salt buVer, 5£ hybridization buVer, and RPA digestion buVer should all be made fresh after 6 months of storage. The SDS present in the stop buVer will precipitate out of solution at room temperature and must be incubated at 37 °C to get it back into solution. Creatine phosphate should be stored at ¡80 °C in 50 L aliquots to avoid multiple freeze/thaw cycles. 2.3. Reaction conditions In vitro transcription-splicing reactions are carried out in a total volume of 25 L. First, 14.5 L of HeLa nuclear extract in buVer D (20 mM Hepes (pH 7.9), 20% glycerol, 0.2 mM EDTA, 0.1 M KCl, and 0.5 mM DTT) [24,25] (14 mg protein/mL) is added to 1.0 L of linearized DNA template (1.0 g/L). The reactions are supplemented to a Wnal concentration of 2 mM MgCl2 · 6H2O, 50 M ZnCl2, and 0.5 U/ L of RNasin Plus RNase inhibitor (Promega) by the addition of 1.0 L MgCl2/ZnCl2 mix and 1.0 L of 12.5 U/ L of RNasin Plus RNase inhibitor to yield a reaction volume of 17.5 L. This mixture is then preincubated at 30 °C for 30 min. We have found that the preincubation step is absolutely necessary for the production of fully spliced product. Following preincubation, the reaction is supplemented to a Wnal concentration of 300 ng poly(dI):poly(dC), 250 ng poly(I):poly(C), 290 M DTT, 10 mM creatine phosphate, 116 M EDTA, 1.0 mM ATP, 600 M GTP, CTP, and UTP, 25 Ci of [-32P]UTP (3000 Ci/mmol) (ICN) with 5.0 L of mix A, and 2.5 L of [-32P]UTP. Reactions are further incubated at 30 °C for up to 3.0 h. HeLa nuclear extract preparation has been described in detail [25] (see also article by Grabowski in this issue). Aliquots of HeLa nuclear extracts are frozen at ¡80 °C and are used only 2–3 times to avoid loss of activity due to multiple freeze/thaw cycles. In addition, the extracts are not precleared by centrifugation to clear of aggregates as this has been observed to decrease splicing eYciency. The human -globin transcripts are approximately 2000 nucleotides in length. We have observed that limiting the concentration of the labeled nucleotide to make transcripts with higher speciWc activity results in a decrease in the number of full length transcripts. Nucleotide concentrations should be optimized for diVerent templates and the conditions described here should serve only as a guide. Furthermore, it is important to note that others have had success with the development of in vitro coupled transcription-splicing systems using diVerent conditions ([20,26], Hicks, Yang, and Hertel personal communication). The reactions are stopped by the addition of 100 L of stop buVer and 50 L of high salt buVer. Samples are extracted once with phenol/chloroform/isoamyl alcohol (25:24:1) and subsequently extracted with chloroform. The extracted RNA is then precipitated using 50 L of 7.5 M ammonium acetate and 600 L of cold 100% ethanol. Ten micrograms of yeast tRNA is added to facilitate precipitation. The samples are then transferred to dry ice for at least 30 min and pelleted at 14,000 rpms for 15 min
at 4 °C. The samples are washed in chilled 80% ethanol, dried, and resuspended in 16 L nuclease-free water. Do not overdry the RNA pellets as they will be very diYcult to resuspend. The method by which we analyze the splicing eYciency of the coupled system is by comparing it to an uncoupled system. One way to analyze uncoupled transcription and splicing is by synthesizing -globin mRNAs using T7 RNAP, purifying those transcripts, and adding those transcripts into the transcription-splicing reactions after the preincubation step. T7 RNAP transcription reactions are carried out in a total volume of 25 L. Reactions contain 400 M ATP, CTP, GTP, and UTP; 25Ci of [32 P]UTP (3000 Ci/mmol), 1.0 g of linearized DNA (digested and puriWed identically to the method described for the coupled reactions); 4 U RNasin Plus RNase inhibitor, 20 U of T7 RNA polymerase Plus (Ambion), and transcription buVer (supplied with the polymerase). Please note that to avoid precipitation of the template DNA, the transcription reaction must be set up at room temperature and the transcription buVer must be added after the water and the nucleotides are already mixed. T7 -globin transcription reactions are incubated at 37 °C for at least 2 h. Transcription reactions are further incubated at 37 °C for an additional 15 min after the addition of 2 L of TURBO DNase (Ambion). Reactions are stopped by the addition of 100 L stop buVer and 50 L high salt buVer; and then are extracted once with phenol/chloroform/isoamyl alcohol and once with chloroform identically to the method already described (see above). For a detailed description of in vitro splicing reactions see article by Hicks et al. in this issue. Another method to study uncoupled transcription and splicing is by adding T7 RNAP directly to the transcription-splicing reaction so that transcripts are synthesized by T7 RNAP in the extract. In these experiments, we dilute the T7 RNAP in buVer D to a Wnal concentration of 0.13 U/L and add 1.0 L of the diluted T7 RNAP to the reaction prior to the preincubation step. The diluted T7 RNAP must be made fresh every time the experiment is performed due to loss of activity caused by freeze/thawing. The remainder of the experiment is completed as described above. 2.4. DNase treatment Once the samples are resuspended in nuclease-free water, they are then DNase treated using TURBO DNase (Ambion) in a total volume of 20 L for at least 1.0 h at 37 °C. The DNase treatment is stopped by the addition of 100 L stop buVer and 50 L high salt buVer, and the samples are phenol/chloroform/isoamyl alcohol extracted, chloroform extracted, and ethanol precipitated with ammonium acetate as described above. We have found that adding DNase directly to the in vitro transcriptionsplicing reaction does not eVectively eliminate DNA contamination, possibly because of the EDTA in the reactions.
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Fig. 2. Expected results from a reverse RNase protection. A proWle of bands from a representative gel is shown on the left. Hybridizations of the probe to the various products resulting from a transcription-splicing reaction are pictured on the right. Both spliced and unspliced products are observed using the reverse RNase protection.
2.5. Analysis of in vitro transcribed-spliced RNA by reverse RNase protection1 Following the DNase treatment and ethanol precipitation, samples are spun at 14,000 rpms, and washed with chilled 80% ethanol, dried, and resuspended in 10 l of 1£ hybridization buVer (40 mM Pipes, pH 6.4, 400 mM NaCl, and 1 mM EDTA). The antisense probe for the reverse RNase protection is transcribed in a total volume of 25 L. Reactions contain 400 M ATP, CTP, GTP, and UTP; 1.0 g of linearized probe DNA; 4 U RNasin Plus RNase inhibitor, 20 U of T3 RNA polymerase Plus (Ambion), and transcription buVer (supplied with the polymerase). The probe is transcribed for 1.5 h at 37 °C. Two microliters of TURBO DNase is added directly to the reaction and the reaction is incubated at 37 °C for an additional 15 min. Reactions are stopped and transcripts are puriWed as indicated above. The probe is 1 It may also be possible to use a conventional RNase protection instead of the reverse protection. We decided to use the reverse protection when an artifact appearing as fully spliced product could not be eliminated with the conventional RNase protection. We believe that this artifact was the result of the exons of the unspliced transcript hybridizing with the probe and the -globin transcript introns looping out. In a reverse RNase protection, a product representing a fully spliced product can only appear when fully spliced product is present because it is the unspliced transcript that is radioactive, not the fully spliced probe.
resuspended in 50 L of 1£ hybridization buVer and 2 l of probe (»0.7 g) per reaction is suYcient to be in probe excess. Hybridization reactions (total volume of 20 L) are set up in PCR tubes and contain the following: 10 L of globin RNA resuspended in 1£ hybridization buVer, 2 L of probe, and an additional 8 L of 1£ hybridization buVer. RNAs and the antisense probe are heated to 95 °C for 5 min to denature the RNAs followed by an overnight incubation at 65 °C. The hybridization is performed in a PCR machine and the ramping rate from 95 to 65 °C is 2.0 °C/s. The following day samples are transferred back to 1.5 mL eppendorf tubes and are RNase digested. A mix of 150 L of RPA digestion buVer combined with 1.5 L of RNase A/T1 Cocktail (Ambion) is added to the hybridized RNAs, and is incubated at 37 °C for 1 h. SDS and Proteinase K (Ambion) are added to a Wnal concentration of 0.5% and 0.275 g/L, respectively, and the samples are incubated at 37 °C for another 15 min. The samples are then phenol/chloroform/isoamyl alcohol extracted, chloroform extracted, and ethanol precipitated using 40 g of tRNA as carrier. Samples are left on dry ice for at least 30 min then pelleted at 14,000 rpm at 4 °C, washed in 80% ethanol, dried, and resuspended in 6 l of 98% formamide buVer. Samples are heated to 100 °C for 3 min and then spun at 14,000 rpms for 1 min before loading. Samples are resolved on 0.4 mm thick denaturing 10% polyacrylamide/8 M urea gels at constant power (50 watts). Glass
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plates are 16.5 cm wide £ 27 cm height £ 3/16 inch thick. It is important to keep in mind that RNase protections should be optimized for the sample of interest and these conditions should only be used as guidelines.
at 30 °C, rather than 37 °C and that we observe a signiWcant amount of transcription that occurs at 25 °C.
2.6. QuantiWcation and analysis of results
4.1. Using the in vitro transcription-splicing system to study splicing eYciency of constitutively spliced -globin transcripts synthesized by RNAP II versus transcripts synthesized by T7 RNAP
We detect several bands using reverse RNase protection (Fig. 2). The two slowest migrating bands (expected to be 445 and 312 nucleotides long) correspond to the 1 + 2 + 3 fully spliced product and 1 + 2 partially spliced product. This was determined by their size, and by cloning and sequencing. We rarely detect 2 + 3 partially spliced product, which retains intron 1. In addition, we observe bands that correspond to RNAs of 90, 222, and 130 nucleotides and which represent protection by exons 1, 2, and 3, respectively. Exons 1 and 3 are always seen as doublets and we believe that this is due to sequence redundancy at the splice sites as well as breathing of the probe. Using reverse RNase protection, we can quantify the levels of the spliced forms of the -globin transcript. Results are analyzed and quantiWed using a Storm Phosphorimager and ImageQuant software (Molecular Dynamics). We calculate splicing eYciency as the ratio of moles of fully spliced transcripts (1 + 2 + 3) divided by moles of all transcripts that yield protection of the probe over exon 3, which includes fully spliced, partially spliced products, and unspliced RNAs: [(1 + 2 + 3)/(1 + 2 + 3) + 3]. We use the moles of exon 3 in our calculations because not all transcripts made by RNAP II are full length transcripts and those not including exon 3 are not substrates for the full splicing reaction. The splicing eYciency {(1 + 2 + 3)/[(1 + 2 + 3) + 3] £ 100} of fully spliced product is consistently between 6 and 10%, although recently higher levels have been achieved by increasing ATP concentrations (data not shown).
4. Applications of the system
Fig. 4A depicts a time course study of -globin transcription splicing analyzed by reverse RNase protection. In
3. Optimization of conditions 3.1. Salt optimum We have determined that a 2 mM MgCl2 concentration is optimal for our system. This is in agreement with previous in vitro splicing studies that also use a MgCl2 concentration of between 1.5 and 2.5mM [27]. In contrast, Hertel and colleagues have observed splicing using 4mM MgCl2 (personal communication). In addition, we have found that addition of 50M ZnCl2 enhances the splicing eYciency in our system, as has been previously demonstrated to improve coupled 3⬘ end formation [18]. The salt optimum yielding the highest splicing eYciency should be determined for diVerent templates. 3.2. Temperature optimum We have tested the eVect of temperature on our system and have concluded that 30 °C is the optimal temperature for the highest splicing eYciency of fully spliced product (Fig. 3). It is interesting to note that our reactions work best
Fig. 3. The eVect of temperature on coupled transcription splicing. Varying temperatures were tested for eVects on splicing. Of those temperatures tested, these data indicate that 30 °C works best for coupled transcription splicing. The lanes labeled with 1 + 2, 2 + 3, and 1 + 2 + 3 have bands that correspond to the indicated spliced products.
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B
Fig. 4. Constitutive splicing using the coupled transcription-splicing assay. A time course study of human -globin transcribed by RNAP II compared with identical transcripts presynthesized by T7 RNAP. (A) Transcripts synthesized by RNAP II are spliced much more eYciently than those transcripts presynthesized by T7 RNAP which are introduced into the reaction at time zero. (B) Graphical representation of the data shown in (A).
the left panel, RNAP II driven -globin transcripts were spliced over a time course of 3 h. We detected the accumulation of transcripts by 15 min and the appearance of fully spliced product by 45 min. A time course with shorter time points revealed that full length transcripts started to accumulate by 5.0 min (data not shown). The right panel of Fig. 4A is a time course study of a presynthesized T7 -globin transcript that was added exogenously as a full length transcript after the preincubation step, very similar to a
conventional in vitro splicing reaction. The quantiWcation reveals that the splicing eYciency of the -globin transcripts synthesized by RNAP II was much greater than the splicing eYciency of the -globin transcripts presynthesized by T7 RNAP. We interpret the greater splicing eYciency in the RNAP II lanes as a result of the functional coupling between transcription and splicing. As depicted in the graph (Fig. 4B), the transcripts presynthesized by T7 RNAP appeared to undergo a much longer lag in splicing
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B
Fig. 5. Constitutive splicing using the coupled transcription-splicing system. A time course analysis of human -globin transcribed by RNAP II compared with identical transcripts synthesized by T7 RNAP in the extract. (A) Transcripts synthesized by RNAP II are at least 100 times more eYciently spliced than those transcripts synthesized by T7 RNAP in the extract. (B) Graphical representation of the data shown in (A).
compared to those transcripts synthesized by RNAP II. This observation strongly suggests that transcription and splicing are coupled processes when transcripts are synthesized by RNAP II. In addition, we observed a preferred order of intron removal. Our data demonstrate that intron 1 was removed Wrst as indicated by the appearance of 1 + 2 spliced product. This result is in agreement with in vivo
data that illustrates that intron 1 is preferentially removed Wrst [28]. In addition to comparing the splicing eYciencies of RNAP II transcripts with presynthesized T7 transcripts, we assessed the splicing eYciencies of RNAP II transcripts with transcripts synthesized by T7 RNAP in the extract. We measured the splicing eYciencies of the transcription-
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A
B
Fig. 6. Alternative splicing using the coupled transcription-splicing system. (A) RT-PCR analysis of in vivo alternative splicing of the FGFR2 minigene construct in triplicate. HeLa S3 cells were transfected with either pI12IIIb-WT or pI12IIIb-UISS/ICE DNA and harvested at 24 h. Two major bands appear, representing IIIb inclusion and IIIb repression (skipping). (B) RT-PCR analysis of the FGFR2 minigene constructs tested in vitro using the coupled transcription-splicing system in duplicate. Here, we observe IIIb inclusion and skipping as seen in vivo as well as an additional IIIb inclusion band that contains intronic sequence (U-intron-IIIb-D).
splicing reactions by performing a time course (Fig. 5A). In the left panel as before, -globin transcripts were synthesized and spliced by RNAP II. Transcripts accumulated as early as 15 min and spliced product appeared by 45 min. In the right panel, T7 RNAP was added exogenously to the transcription-splicing reactions. T7 RNAP transcribed to approximately the same levels as RNAP II, but spliced products did not accumulate (Fig. 5B). The inability of transcripts synthesized by T7 RNAP to splice may be due to multiple reasons. First, T7 transcripts may be unable to fold into the proper conformation necessary for the eYcient binding of splicing factors. Second, the T7 synthesized transcripts may form R-loops with the DNA template, which would then inhibit binding of splicing factors. And last, the the T7 synthesized transcripts may preferentially form inactive mRNPs. In agreement with our data, Hertel and colleagues have independently observed a decrease in splicing eYciency when transcripts are synthesized by T7 RNAP in the extract compared to transcripts synthesized by RNAP II (personal communication).
We also analyzed mutations of the 5⬘ splice sites (SS) of exons 1 and 2 individually. Mutating the 5⬘SS of exon 1 yielded an increase in 2 + 3 splicing and activation of a cryptic splice site; whereas, interestingly, mutating the 5⬘SS of exon 2 lead to a dramatic decrease in 1 + 2 and 1 + 2+ 3 splicing (data not shown). This latter result is highly suggestive of exon deWnition. If exon deWnition is occurring, we would expect to see exon skipping and indeed, using RTPCR, we observed increased exon skipping in the 5⬘SS exon 2 mutant compared to the wild-type -globin construct (data not shown). 4.2. Using the in vitro transcription-splicing system to study splicing eYciency of alternatively spliced FGFR2 minigene transcripts synthesized by RNAP II versus transcripts synthesized by T7 RNAP FGFR2 undergoes a mutually exclusive alternative splicing event where the FGFR2 IIIb isoform is expressed in epithelial cells and IIIc isoform is expressed in mesen-
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chymal cells. A critical aspect of this regulation is the silencing of exon IIIb by exonic and intronic splicing silencers [29]. The critical intronic silencers are found both upstream (UISS) and downstream (ICE) of exon IIIb. We tested two constructs in our system, pI12IIIb-WT transcripts, which contain both silencers, and pI12IIIbUISS/ICE RNAs, where UISS and ICE have been deleted. We wanted to see whether our in vitro transcription-splicing system would recapitulate in vivo data. When these constructs are transiently transfected into HeLa cells, exon IIIb is silenced in the pI12IIIb-WT transcripts resulting in mostly skipped product, whereas exon IIIb is included in pI12IIIb-UISS/ICE RNAs (Fig. 6A). When we put these constructs into our in vitro transcription-splicing system, we observed similar results (Fig. 6B). In fact, our system not only recapitulates what happens in vivo, but also seems to be a superior method to study alternative splicing compared to conventional in vitro splicing methods using presynthesized T7 transcripts. More speciWcally, the ratio of properly spliced to improperly spliced IIIb inclusion products is signiWcantly higher when transcripts are synthesized by RNAP II compared with presynthesized T7 transcripts. These data suggest that the transcription-splicing system described here should be amenable to study alternative splicing decisions in vitro. 5. Concluding remarks We have developed an in vitro system to study the functional coupling of transcription and splicing using HeLa nuclear extracts that is capable of accurate and eYcient splicing of three and four exon constructs (here and data not shown). Here, we demonstrate that human -globin pre-mRNAs driven by RNAP II splice more eYciently than those presynthesized by T7 RNAP. Furthermore, transcripts synthesized by T7 RNAP in the extract have very low splicing eYciency. Additionally, we have demonstrated that our system can be used to study alternative splicing, more speciWcally FGFR2 exon IIIb silencing. Overall, the results presented in this article demonstrate that the in vitro transcription-splicing system described provides an important alternative to conventional in vitro methods used to study pre-mRNA splicing. Ultimately, we hope that this system, which more closely resembles what occurs in vivo, will be widely used as a method to study both constitutive and alternative splicing.
Acknowledgments We thank all members of the Garcia-Blanco laboratory, especially James Pearson, for all the invaluable advice and helpful suggestions and Klemens Hertel for generously sharing data before publication and for his comments on this article. We thank Bruce Sullenger and Bryan Cullen for their generous gifts of the -globin plasmids. This work was funded by NIH Grant RO1 GM071037 to M.G.B. References [1] [2] [3] [4] [5]
[6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
[26] [27] [28] [29]
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In vitro coupled transcription splicing Barbara J. Natalizio a, Mariano A. Garcia-Blanco a,b,¤ a
Departments of Molecular Genetics and Microbiology, Center for RNA Biology, Duke University Medical Center, Durham, NC 27710, USA b Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA Accepted 21 July 2005
Abstract Many convincing studies published in recent years strongly support coupling of transcription and pre-mRNA processing. Despite key advances in our understanding of these processes, there is a lack of a robust in vitro system in which to study the mechanism of this coupling for complex pre-mRNAs. Here, we describe an in vitro system capable of transcribing and splicing complex transcripts with three and four exons. We also demonstrate how the system can be used to study exon silencing in vitro. We believe that this system will be a useful tool to study the mechanisms that mediate the coupling of transcription and pre-mRNA processing. 2005 Elsevier Inc. All rights reserved. Keywords: Coupling; Cotranscriptional splicing; mRNA processing; RNA polymerase II; T7 RNA polymerase; Transcription
1. Introduction The occurrence of cotranscriptional mRNA processing is widely accepted as the cytological, biochemical, and functional evidence supporting it continues to expand. The Wrst demonstration of cotranscriptional splicing came from the direct visualization of Miller chromatin spreads from Drosophila melanogaster embryos [1]. Further evidence in support of cotranscriptional splicing resulted from examining the splicing rate of transcripts from the Balbani ring 1 gene in Chironomus tentans and from the human dystrophin gene [2,3]. These studies demonstrated that while 5⬘ introns are mostly removed cotranscriptionally, the 3⬘ introns of these long transcripts are generally removed posttranscriptionally. Cotranscriptional pre-mRNA processing depends on speciWc components of RNA polymerase II (RNAP II) [4] and requires the carboxy-terminal domain (CTD) of the largest RNAP II subunit [5]. Studies focusing on the role of the CTD in processing emerged shortly after Greenleaf [6] proposed that the highly charged CTD may *
Corresponding author. Fax: +1 919 613 8646. E-mail address: [email protected] (M.A. Garcia-Blanco).
1046-2023/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2005.07.013
serve as a scaVold for pre-mRNA processing factors (reviewed in [7]). Truncation of the CTD speciWcally inhibits capping, splicing of some introns, cleavage/polyadenylation, and transcription termination (reviewed in [8–12]). Currently, most biochemical studies of pre-mRNA processing are carried out independent of transcription. Processing precursors are synthesized by bacteriophage RNA polymerases (e.g., T7 RNAP), puriWed, and subsequently incubated with cellular extracts [13–17]. The hope is that establishing a system whereby transcription and premRNA processing are functionally and perhaps, physically coupled will help elucidate connections between transcription and splicing that are currently diYcult to study by conventional in vitro pre-mRNA processing systems. To this end, we and others have developed in vitro systems to study coupling of transcription with 3⬘ end formation and polyadenylation [18,19] or with splicing of single intron premRNAs [20]. Although the latter could eYciently transcribe and splice single intron transcripts, it did not eVectively handle more complex splicing precursors that are most likely impacted by cotranscriptional coupling. With this in mind, we have developed a system that can splice larger and more complex transcripts, including three and
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four exon constructs. Our system can properly and eYciently transcribe, and splice both constitutively spliced transcripts as well as alternatively spliced transcripts. Furthermore, this system can recapitulate exon deWnition. This assay has also been used to examine trans-splicing and we observe that the delay in synthesis of the 3⬘ splice site facilitates the trans-splicing of the upstream 5⬘ splice site, as has been observed in vivo [21]. In addition, using time course analysis we can examine the order of intron removal. Moreover, we have data that are consistent with increased stability of RNAP II transcripts versus almost identical T7 RNAP transcripts. This phenomenon has been carefully documented by Hertel and colleagues also using an in vitro system (personal communication Hicks, Yang, and Hertel). This protection of the RNAP II transcripts from nuclear degradation may be attributed to the coupling of transcription and splicing. We believe that the system described here will serve as a valuable resource for studying mRNA processing in the context of coupling.
plates cleaved immediately downstream of the 3⬘UTR. Complete digestion and purity of the DNA template are imperative to the success of the downstream applications. Following digestion, templates are phenol/chloroform/isoamyl alcohol (25:24:1) extracted once, chloroform extracted twice, and ethanol precipitated with 1:10 vol of 3 M sodium acetate, pH 7.0. They are then pelleted at 14,000 rpms, washed once with chilled 80% ethanol, dried, and resuspended in nuclease-free water (Ambion) to a Wnal concentration of 1.0 g/L. Please note that we do not typically gel purify the DNA fragments of interest. Although PCR templates can be used in lieu of restriction fragments, in our experience these PCR templates do not perform as well as linearized plasmids. It is important to note that any plasmid that is normally used to express a splicing precursor in cells can be used for these in vitro reactions.
2. Description of method
Aqueous buVers and solutions are all made with nuclease-free water and are not DEPC treated.
2.2. BuVers and solutions
2.1. Plasmids/vectors DNA templates for the in vitro transcription-splicing reactions were constructed by inserting the complete human -globin gene, including both the 5⬘ and 3⬘ untranslated regions (UTRs) without the endogenous globin promoter, into pBC12/CMV [22,23] (Fig. 1). An identical -globin construct driven by T7 RNAP was created by removing the CMV promoter region and replacing it with the T7 promoter region. Additionally, a 15 nucleotide sequence devoid of pyrimidines was added to both constructs at the transcription initiation start site to prevent the polymerase from prematurely terminating. In vitro transcription-splicing reactions have also been performed with templates cloned into pcDNA3 where both the CMV promoter and T7 promoter are present in one vector and these reactions seem to work equally as well (data not shown). The -globin templates were digested both upstream and downstream of the region of interest with BsrB1 restriction endonuclease to prevent transcription from upstream cryptic promoters. This digestion cleaves approximately 400 nucleotides downstream of the 3⬘UTR of -globin. We have observed that linearizing the DNA template at this distance downstream of the 3⬘UTR results in increased splicing eYciency compared to tem-
NTP mix: 25 mM ATP and 15 mM CTP, GTP, UTP high purity nucleotides for RNA synthesis (Amersham Biosciences) (store at ¡20 °C). Mix A: 5 mM ATP and 3 mM CTP, GTP, UTP; 0.05 g/ L poly(I):poly(C) (Amersham Biosciences); 0.06 g/L poly(dI):poly(dC) (Amersham Biosciences); 50 mM creatine phosphate (Sigma Cat# P7936); 0.5 mM DTT (store at ¡20 °C). MgCl2//ZnCl2 mix: 50 mM MgCl2 · 6H2O (EMD); 1.25 mM ZnCl2 (CalBiochem) (store at ¡20 °C). Stop buVer: 100 mM Tris–HCl (pH 7.5), 200 mM NaCl, 10 mM EDTA, 300 mM NaOAc, 0.75% SDS, 0.025 mg/ mL yeast tRNA (Sigma Cat# R5636) (store at 25 °C). High salt buVer: 10 mM Tris–HCl (pH 7.5), 50 mM EDTA, 100 mM LiCl, 0.5% SDS, 7.0 M urea (store at 25 °C), 7.5 M NH4 OAc (store at 25 °C). 5£ RPA hybridization buVer: 200 mM Pipes (pH 6.4), 2.0 M NaCl, 5.0 mM EDTA (store at ¡20 °C). Hybridization buVer is diluted with formamide. RPA digestion buVer: 300 mM NaCl, 10 mM Tris–HCl (pH 7.5), 5.0 mM EDTA (store at ¡20 °C). Formamide buVer: 95% formamide, 18 mM EDTA, 0.025% SDS, xylene cyanol, and bromophenol blue (store at ¡20 °C).
Fig. 1. Schematic representation of human -globin construct. Human -globin is transcribed by either RNAP II or T7 polymerase using the CMV-IE or T7 promoter, respectively. The shaded boxes represent non--globin sequence. A pyrimidine free region is inserted at the transcription start site. The last 422 nucleotides of the construct are pBC12 vector sequence. The position of the poly A site is indicated by the black box within exon 3.
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Please note that the NTP mix and mix A should not be kept for longer than one month. Furthermore, stop buVer, high salt buVer, 5£ hybridization buVer, and RPA digestion buVer should all be made fresh after 6 months of storage. The SDS present in the stop buVer will precipitate out of solution at room temperature and must be incubated at 37 °C to get it back into solution. Creatine phosphate should be stored at ¡80 °C in 50 L aliquots to avoid multiple freeze/thaw cycles. 2.3. Reaction conditions In vitro transcription-splicing reactions are carried out in a total volume of 25 L. First, 14.5 L of HeLa nuclear extract in buVer D (20 mM Hepes (pH 7.9), 20% glycerol, 0.2 mM EDTA, 0.1 M KCl, and 0.5 mM DTT) [24,25] (14 mg protein/mL) is added to 1.0 L of linearized DNA template (1.0 g/L). The reactions are supplemented to a Wnal concentration of 2 mM MgCl2 · 6H2O, 50 M ZnCl2, and 0.5 U/ L of RNasin Plus RNase inhibitor (Promega) by the addition of 1.0 L MgCl2/ZnCl2 mix and 1.0 L of 12.5 U/ L of RNasin Plus RNase inhibitor to yield a reaction volume of 17.5 L. This mixture is then preincubated at 30 °C for 30 min. We have found that the preincubation step is absolutely necessary for the production of fully spliced product. Following preincubation, the reaction is supplemented to a Wnal concentration of 300 ng poly(dI):poly(dC), 250 ng poly(I):poly(C), 290 M DTT, 10 mM creatine phosphate, 116 M EDTA, 1.0 mM ATP, 600 M GTP, CTP, and UTP, 25 Ci of [-32P]UTP (3000 Ci/mmol) (ICN) with 5.0 L of mix A, and 2.5 L of [-32P]UTP. Reactions are further incubated at 30 °C for up to 3.0 h. HeLa nuclear extract preparation has been described in detail [25] (see also article by Grabowski in this issue). Aliquots of HeLa nuclear extracts are frozen at ¡80 °C and are used only 2–3 times to avoid loss of activity due to multiple freeze/thaw cycles. In addition, the extracts are not precleared by centrifugation to clear of aggregates as this has been observed to decrease splicing eYciency. The human -globin transcripts are approximately 2000 nucleotides in length. We have observed that limiting the concentration of the labeled nucleotide to make transcripts with higher speciWc activity results in a decrease in the number of full length transcripts. Nucleotide concentrations should be optimized for diVerent templates and the conditions described here should serve only as a guide. Furthermore, it is important to note that others have had success with the development of in vitro coupled transcription-splicing systems using diVerent conditions ([20,26], Hicks, Yang, and Hertel personal communication). The reactions are stopped by the addition of 100 L of stop buVer and 50 L of high salt buVer. Samples are extracted once with phenol/chloroform/isoamyl alcohol (25:24:1) and subsequently extracted with chloroform. The extracted RNA is then precipitated using 50 L of 7.5 M ammonium acetate and 600 L of cold 100% ethanol. Ten micrograms of yeast tRNA is added to facilitate precipitation. The samples are then transferred to dry ice for at least 30 min and pelleted at 14,000 rpms for 15 min
at 4 °C. The samples are washed in chilled 80% ethanol, dried, and resuspended in 16 L nuclease-free water. Do not overdry the RNA pellets as they will be very diYcult to resuspend. The method by which we analyze the splicing eYciency of the coupled system is by comparing it to an uncoupled system. One way to analyze uncoupled transcription and splicing is by synthesizing -globin mRNAs using T7 RNAP, purifying those transcripts, and adding those transcripts into the transcription-splicing reactions after the preincubation step. T7 RNAP transcription reactions are carried out in a total volume of 25 L. Reactions contain 400 M ATP, CTP, GTP, and UTP; 25Ci of [32 P]UTP (3000 Ci/mmol), 1.0 g of linearized DNA (digested and puriWed identically to the method described for the coupled reactions); 4 U RNasin Plus RNase inhibitor, 20 U of T7 RNA polymerase Plus (Ambion), and transcription buVer (supplied with the polymerase). Please note that to avoid precipitation of the template DNA, the transcription reaction must be set up at room temperature and the transcription buVer must be added after the water and the nucleotides are already mixed. T7 -globin transcription reactions are incubated at 37 °C for at least 2 h. Transcription reactions are further incubated at 37 °C for an additional 15 min after the addition of 2 L of TURBO DNase (Ambion). Reactions are stopped by the addition of 100 L stop buVer and 50 L high salt buVer; and then are extracted once with phenol/chloroform/isoamyl alcohol and once with chloroform identically to the method already described (see above). For a detailed description of in vitro splicing reactions see article by Hicks et al. in this issue. Another method to study uncoupled transcription and splicing is by adding T7 RNAP directly to the transcription-splicing reaction so that transcripts are synthesized by T7 RNAP in the extract. In these experiments, we dilute the T7 RNAP in buVer D to a Wnal concentration of 0.13 U/L and add 1.0 L of the diluted T7 RNAP to the reaction prior to the preincubation step. The diluted T7 RNAP must be made fresh every time the experiment is performed due to loss of activity caused by freeze/thawing. The remainder of the experiment is completed as described above. 2.4. DNase treatment Once the samples are resuspended in nuclease-free water, they are then DNase treated using TURBO DNase (Ambion) in a total volume of 20 L for at least 1.0 h at 37 °C. The DNase treatment is stopped by the addition of 100 L stop buVer and 50 L high salt buVer, and the samples are phenol/chloroform/isoamyl alcohol extracted, chloroform extracted, and ethanol precipitated with ammonium acetate as described above. We have found that adding DNase directly to the in vitro transcriptionsplicing reaction does not eVectively eliminate DNA contamination, possibly because of the EDTA in the reactions.
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Fig. 2. Expected results from a reverse RNase protection. A proWle of bands from a representative gel is shown on the left. Hybridizations of the probe to the various products resulting from a transcription-splicing reaction are pictured on the right. Both spliced and unspliced products are observed using the reverse RNase protection.
2.5. Analysis of in vitro transcribed-spliced RNA by reverse RNase protection1 Following the DNase treatment and ethanol precipitation, samples are spun at 14,000 rpms, and washed with chilled 80% ethanol, dried, and resuspended in 10 l of 1£ hybridization buVer (40 mM Pipes, pH 6.4, 400 mM NaCl, and 1 mM EDTA). The antisense probe for the reverse RNase protection is transcribed in a total volume of 25 L. Reactions contain 400 M ATP, CTP, GTP, and UTP; 1.0 g of linearized probe DNA; 4 U RNasin Plus RNase inhibitor, 20 U of T3 RNA polymerase Plus (Ambion), and transcription buVer (supplied with the polymerase). The probe is transcribed for 1.5 h at 37 °C. Two microliters of TURBO DNase is added directly to the reaction and the reaction is incubated at 37 °C for an additional 15 min. Reactions are stopped and transcripts are puriWed as indicated above. The probe is 1 It may also be possible to use a conventional RNase protection instead of the reverse protection. We decided to use the reverse protection when an artifact appearing as fully spliced product could not be eliminated with the conventional RNase protection. We believe that this artifact was the result of the exons of the unspliced transcript hybridizing with the probe and the -globin transcript introns looping out. In a reverse RNase protection, a product representing a fully spliced product can only appear when fully spliced product is present because it is the unspliced transcript that is radioactive, not the fully spliced probe.
resuspended in 50 L of 1£ hybridization buVer and 2 l of probe (»0.7 g) per reaction is suYcient to be in probe excess. Hybridization reactions (total volume of 20 L) are set up in PCR tubes and contain the following: 10 L of globin RNA resuspended in 1£ hybridization buVer, 2 L of probe, and an additional 8 L of 1£ hybridization buVer. RNAs and the antisense probe are heated to 95 °C for 5 min to denature the RNAs followed by an overnight incubation at 65 °C. The hybridization is performed in a PCR machine and the ramping rate from 95 to 65 °C is 2.0 °C/s. The following day samples are transferred back to 1.5 mL eppendorf tubes and are RNase digested. A mix of 150 L of RPA digestion buVer combined with 1.5 L of RNase A/T1 Cocktail (Ambion) is added to the hybridized RNAs, and is incubated at 37 °C for 1 h. SDS and Proteinase K (Ambion) are added to a Wnal concentration of 0.5% and 0.275 g/L, respectively, and the samples are incubated at 37 °C for another 15 min. The samples are then phenol/chloroform/isoamyl alcohol extracted, chloroform extracted, and ethanol precipitated using 40 g of tRNA as carrier. Samples are left on dry ice for at least 30 min then pelleted at 14,000 rpm at 4 °C, washed in 80% ethanol, dried, and resuspended in 6 l of 98% formamide buVer. Samples are heated to 100 °C for 3 min and then spun at 14,000 rpms for 1 min before loading. Samples are resolved on 0.4 mm thick denaturing 10% polyacrylamide/8 M urea gels at constant power (50 watts). Glass
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plates are 16.5 cm wide £ 27 cm height £ 3/16 inch thick. It is important to keep in mind that RNase protections should be optimized for the sample of interest and these conditions should only be used as guidelines.
at 30 °C, rather than 37 °C and that we observe a signiWcant amount of transcription that occurs at 25 °C.
2.6. QuantiWcation and analysis of results
4.1. Using the in vitro transcription-splicing system to study splicing eYciency of constitutively spliced -globin transcripts synthesized by RNAP II versus transcripts synthesized by T7 RNAP
We detect several bands using reverse RNase protection (Fig. 2). The two slowest migrating bands (expected to be 445 and 312 nucleotides long) correspond to the 1 + 2 + 3 fully spliced product and 1 + 2 partially spliced product. This was determined by their size, and by cloning and sequencing. We rarely detect 2 + 3 partially spliced product, which retains intron 1. In addition, we observe bands that correspond to RNAs of 90, 222, and 130 nucleotides and which represent protection by exons 1, 2, and 3, respectively. Exons 1 and 3 are always seen as doublets and we believe that this is due to sequence redundancy at the splice sites as well as breathing of the probe. Using reverse RNase protection, we can quantify the levels of the spliced forms of the -globin transcript. Results are analyzed and quantiWed using a Storm Phosphorimager and ImageQuant software (Molecular Dynamics). We calculate splicing eYciency as the ratio of moles of fully spliced transcripts (1 + 2 + 3) divided by moles of all transcripts that yield protection of the probe over exon 3, which includes fully spliced, partially spliced products, and unspliced RNAs: [(1 + 2 + 3)/(1 + 2 + 3) + 3]. We use the moles of exon 3 in our calculations because not all transcripts made by RNAP II are full length transcripts and those not including exon 3 are not substrates for the full splicing reaction. The splicing eYciency {(1 + 2 + 3)/[(1 + 2 + 3) + 3] £ 100} of fully spliced product is consistently between 6 and 10%, although recently higher levels have been achieved by increasing ATP concentrations (data not shown).
4. Applications of the system
Fig. 4A depicts a time course study of -globin transcription splicing analyzed by reverse RNase protection. In
3. Optimization of conditions 3.1. Salt optimum We have determined that a 2 mM MgCl2 concentration is optimal for our system. This is in agreement with previous in vitro splicing studies that also use a MgCl2 concentration of between 1.5 and 2.5mM [27]. In contrast, Hertel and colleagues have observed splicing using 4mM MgCl2 (personal communication). In addition, we have found that addition of 50M ZnCl2 enhances the splicing eYciency in our system, as has been previously demonstrated to improve coupled 3⬘ end formation [18]. The salt optimum yielding the highest splicing eYciency should be determined for diVerent templates. 3.2. Temperature optimum We have tested the eVect of temperature on our system and have concluded that 30 °C is the optimal temperature for the highest splicing eYciency of fully spliced product (Fig. 3). It is interesting to note that our reactions work best
Fig. 3. The eVect of temperature on coupled transcription splicing. Varying temperatures were tested for eVects on splicing. Of those temperatures tested, these data indicate that 30 °C works best for coupled transcription splicing. The lanes labeled with 1 + 2, 2 + 3, and 1 + 2 + 3 have bands that correspond to the indicated spliced products.
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B
Fig. 4. Constitutive splicing using the coupled transcription-splicing assay. A time course study of human -globin transcribed by RNAP II compared with identical transcripts presynthesized by T7 RNAP. (A) Transcripts synthesized by RNAP II are spliced much more eYciently than those transcripts presynthesized by T7 RNAP which are introduced into the reaction at time zero. (B) Graphical representation of the data shown in (A).
the left panel, RNAP II driven -globin transcripts were spliced over a time course of 3 h. We detected the accumulation of transcripts by 15 min and the appearance of fully spliced product by 45 min. A time course with shorter time points revealed that full length transcripts started to accumulate by 5.0 min (data not shown). The right panel of Fig. 4A is a time course study of a presynthesized T7 -globin transcript that was added exogenously as a full length transcript after the preincubation step, very similar to a
conventional in vitro splicing reaction. The quantiWcation reveals that the splicing eYciency of the -globin transcripts synthesized by RNAP II was much greater than the splicing eYciency of the -globin transcripts presynthesized by T7 RNAP. We interpret the greater splicing eYciency in the RNAP II lanes as a result of the functional coupling between transcription and splicing. As depicted in the graph (Fig. 4B), the transcripts presynthesized by T7 RNAP appeared to undergo a much longer lag in splicing
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B
Fig. 5. Constitutive splicing using the coupled transcription-splicing system. A time course analysis of human -globin transcribed by RNAP II compared with identical transcripts synthesized by T7 RNAP in the extract. (A) Transcripts synthesized by RNAP II are at least 100 times more eYciently spliced than those transcripts synthesized by T7 RNAP in the extract. (B) Graphical representation of the data shown in (A).
compared to those transcripts synthesized by RNAP II. This observation strongly suggests that transcription and splicing are coupled processes when transcripts are synthesized by RNAP II. In addition, we observed a preferred order of intron removal. Our data demonstrate that intron 1 was removed Wrst as indicated by the appearance of 1 + 2 spliced product. This result is in agreement with in vivo
data that illustrates that intron 1 is preferentially removed Wrst [28]. In addition to comparing the splicing eYciencies of RNAP II transcripts with presynthesized T7 transcripts, we assessed the splicing eYciencies of RNAP II transcripts with transcripts synthesized by T7 RNAP in the extract. We measured the splicing eYciencies of the transcription-
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A
B
Fig. 6. Alternative splicing using the coupled transcription-splicing system. (A) RT-PCR analysis of in vivo alternative splicing of the FGFR2 minigene construct in triplicate. HeLa S3 cells were transfected with either pI12IIIb-WT or pI12IIIb-UISS/ICE DNA and harvested at 24 h. Two major bands appear, representing IIIb inclusion and IIIb repression (skipping). (B) RT-PCR analysis of the FGFR2 minigene constructs tested in vitro using the coupled transcription-splicing system in duplicate. Here, we observe IIIb inclusion and skipping as seen in vivo as well as an additional IIIb inclusion band that contains intronic sequence (U-intron-IIIb-D).
splicing reactions by performing a time course (Fig. 5A). In the left panel as before, -globin transcripts were synthesized and spliced by RNAP II. Transcripts accumulated as early as 15 min and spliced product appeared by 45 min. In the right panel, T7 RNAP was added exogenously to the transcription-splicing reactions. T7 RNAP transcribed to approximately the same levels as RNAP II, but spliced products did not accumulate (Fig. 5B). The inability of transcripts synthesized by T7 RNAP to splice may be due to multiple reasons. First, T7 transcripts may be unable to fold into the proper conformation necessary for the eYcient binding of splicing factors. Second, the T7 synthesized transcripts may form R-loops with the DNA template, which would then inhibit binding of splicing factors. And last, the the T7 synthesized transcripts may preferentially form inactive mRNPs. In agreement with our data, Hertel and colleagues have independently observed a decrease in splicing eYciency when transcripts are synthesized by T7 RNAP in the extract compared to transcripts synthesized by RNAP II (personal communication).
We also analyzed mutations of the 5⬘ splice sites (SS) of exons 1 and 2 individually. Mutating the 5⬘SS of exon 1 yielded an increase in 2 + 3 splicing and activation of a cryptic splice site; whereas, interestingly, mutating the 5⬘SS of exon 2 lead to a dramatic decrease in 1 + 2 and 1 + 2+ 3 splicing (data not shown). This latter result is highly suggestive of exon deWnition. If exon deWnition is occurring, we would expect to see exon skipping and indeed, using RTPCR, we observed increased exon skipping in the 5⬘SS exon 2 mutant compared to the wild-type -globin construct (data not shown). 4.2. Using the in vitro transcription-splicing system to study splicing eYciency of alternatively spliced FGFR2 minigene transcripts synthesized by RNAP II versus transcripts synthesized by T7 RNAP FGFR2 undergoes a mutually exclusive alternative splicing event where the FGFR2 IIIb isoform is expressed in epithelial cells and IIIc isoform is expressed in mesen-
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chymal cells. A critical aspect of this regulation is the silencing of exon IIIb by exonic and intronic splicing silencers [29]. The critical intronic silencers are found both upstream (UISS) and downstream (ICE) of exon IIIb. We tested two constructs in our system, pI12IIIb-WT transcripts, which contain both silencers, and pI12IIIbUISS/ICE RNAs, where UISS and ICE have been deleted. We wanted to see whether our in vitro transcription-splicing system would recapitulate in vivo data. When these constructs are transiently transfected into HeLa cells, exon IIIb is silenced in the pI12IIIb-WT transcripts resulting in mostly skipped product, whereas exon IIIb is included in pI12IIIb-UISS/ICE RNAs (Fig. 6A). When we put these constructs into our in vitro transcription-splicing system, we observed similar results (Fig. 6B). In fact, our system not only recapitulates what happens in vivo, but also seems to be a superior method to study alternative splicing compared to conventional in vitro splicing methods using presynthesized T7 transcripts. More speciWcally, the ratio of properly spliced to improperly spliced IIIb inclusion products is signiWcantly higher when transcripts are synthesized by RNAP II compared with presynthesized T7 transcripts. These data suggest that the transcription-splicing system described here should be amenable to study alternative splicing decisions in vitro. 5. Concluding remarks We have developed an in vitro system to study the functional coupling of transcription and splicing using HeLa nuclear extracts that is capable of accurate and eYcient splicing of three and four exon constructs (here and data not shown). Here, we demonstrate that human -globin pre-mRNAs driven by RNAP II splice more eYciently than those presynthesized by T7 RNAP. Furthermore, transcripts synthesized by T7 RNAP in the extract have very low splicing eYciency. Additionally, we have demonstrated that our system can be used to study alternative splicing, more speciWcally FGFR2 exon IIIb silencing. Overall, the results presented in this article demonstrate that the in vitro transcription-splicing system described provides an important alternative to conventional in vitro methods used to study pre-mRNA splicing. Ultimately, we hope that this system, which more closely resembles what occurs in vivo, will be widely used as a method to study both constitutive and alternative splicing.
Acknowledgments We thank all members of the Garcia-Blanco laboratory, especially James Pearson, for all the invaluable advice and helpful suggestions and Klemens Hertel for generously sharing data before publication and for his comments on this article. We thank Bruce Sullenger and Bryan Cullen for their generous gifts of the -globin plasmids. This work was funded by NIH Grant RO1 GM071037 to M.G.B. References [1] [2] [3] [4] [5]
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