Name: using the e1a minigene tool to study mrna splicing changes
Uploaded: 2021-04-22T13:30:00
Description: Watch this Scientific Journal Video about Using the E1A Minigene Tool to Study mRNA Splicing Changes at JoVE.com

We thank Funda&#231;&#227;o de Amparo a Pesquisa do Estado de S&#227;o Paulo (FAPESP, through Grant Tem&#225;tico 2017/03489-1 to JK and fellowship to FLB 2018/05350-3) and the Conselho Nacional de Desenvolvimento Cientifico e Tecnol&#243;gico (CNPq) for funding this research. We would like to thank Dr Adrian Krainer for providing the pMTE1A plasmid and Zerler and colleagues for their work in E1A cloning. We also thank Prof. Dr. Patr&#237;cia Moriel, Prof. Dr. Wanda Pereira Almeida, Prof. Dr. Marcelo Lancellotti and Prof. Dr. Karina Kogo Cogo M&#252;ller to allow us to use their laboratory space and equipment.

1. Plating cells
NOTE: In this described protocol, HEK293 stable cell lines, previously generated for stable inducible expression of Nek4 were used21, however, the same protocol is suitable to many other cell lines, such as HEK29322, HeLa23,24,25,26, U-2 OS27, COS728, SH-SY5...

<ol>
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Minigenes are important tools to determine the effects in global alternative splicing in vivo. The adenoviral minigene E1A has been used successfully for decades to evaluate the role of proteins by increasing the amount of these in the cell13,14. Here, we propose the minigene E1A use for evaluating alternative splicing after chemotherapeutic exposure. A stable cell line expressing Nek4 isoform 1 was used, avoiding the artifacts of overexpression caused by transie...

Splicing is among the most important steps in eukaryotic mRNA processing that occurs simultaneously to 5&#180;mRNA capping and 3&#180;mRNA polyadenylation, comprising of intron removal followed by exon junction. The recognition of the splicing sites (SS) by the spliceosome, a ribonucleoprotein complex containing small ribonucleoproteins (snRNP U1, U2, U4 and U6), small RNAs (snRNAs) and several regulatory proteins1 is necessary for splicing.
Besides intron removal (constitutive splicing), in eukaryotes, introns can be retained and exons can be excluded, configuring the process called mRNA alternative splicing (AS). The alternative pre-mRNA splicing expands the coding capacity of eukaryotic genomes allowing the production of a large and diverse number of proteins from a relatively small number of genes. It is estimated that 95-100% of human mRNAs that contain more than one exon can undergo alternative splicing2,3. This is fundamental for biological processes like neuronal development, apoptosis activation and cellular stress response4, providing the organism alternatives to regulate cell functioning using the same repertoire of genes.
The machinery necessary for alternative splicing is the same used for constitutive splicing and the usage of the SS is the main determinant for alternative splicing occurrence. Constitutive splicing is related to the use of strong splicing sites, which are usually more similar to consensus motifs for spliceosome recognition5.
Alternative exons are typically recognized less efficiently than constitutive exons once its cis-regulatory elements, the sequences in 5&#180;SS and 3&#180;SS flanking these exons, show an inferior binding capacity to the spliceosome. mRNA also contains regions named enhancers or silencers located in exons (exonic splicing enhancers (ESEs) and exonic splicing silencers (ESSs)) and introns (intronic splicing enhancers (ISEs) and intronic splicing silencers (ISSs)) that enhance or repress exon usage, respectively5. These sequences are recognized by trans-regulatory elements, or splicing factors (SF). SFs are represented mainly by two families of proteins, the serine/arginine rich splicing factors (SRSFs) which bind to ESEs and the family of heterogeneous nuclear ribonucleoproteins (hnRNPs) which bind to ESSs sequences5.
Alternative splicing can be modulated by phosphorylation/ dephosphorylation of trans- factors modifying the interactions partners and cellular localization of splicing factors6,7,8. Identifying new regulators of splicing factors can provide new tools to regulate splicing and, consequently, some cancer treatments.
Anufrieva et al.9, in a mRNA microarray gene expression profile, observed consistent changes in levels of spliceosomal components in 101 cell lines and after different stress conditions (platinum-based drugs, gamma irradiation, topoisomerase inhibitors, tyrosine kinase inhibitors and taxanes). The relationship among splicing pattern and chemotherapy efficacy has already been demonstrated in lung cancer cells, which are chemotherapy resistant, showing changes in caspase-9 variants rate10. HEK293 cells treated with chemotherapeutics panel show changes in splicing with an increase in pro-apoptotic variants. Gabriel et al.11 observed changes in at least 700 events of splicing after cisplatin treatment in different cell lines, pointing out that splicing pathways are cisplatin-affected. Splicing modulators have already demonstrated anti-tumoral activity, showing that splicing is important to tumoral development and, mainly, chemotherapy response12. Hence, characterizing new proteins that regulate splicing after cellular stressors agents, like chemotherapeutics, is very important to discover new strategies of treatment.
The clues of alternative splicing regulation from interactome studies, particularly important to characterize functions of new or uncharacterized proteins, can demand a more general and simple approach to verify the real role of the protein in AS. Minigenes are important tools for analysis of the general role of a protein affecting splicing regulation. They contain segments from a gene of interest containing alternatively spliced and flanking genomic regions13. Using a minigene tool allows the analysis of splicing in vivo with several advantages such as the length of the minigene which is minor and therefore is not a limitation to the amplification reaction; the same minigene can be evaluated in different cell lines; all cellular components, mainly their regulating post-translational modification (phosphorylation and changes in cell compartments) are present and can be addressed13,14. Moreover, changes in alternative splicing pattern can be observed after cellular stress and, the use of a minigene system, allow to identify the pathway being modulated by different stimuli.
There are several minigene systems already described which are specific for different kinds of splicing events13,14, however, as a preliminary assay, the minigene E1A15 is a very well established alternative splicing reporter system for the study of 5&#180;SS selection in vivo. From only one gene, E1A, five mRNAs are produced by alternative splicing based on selection of three different 5&#8242; splice sites and of one major or one minor 3&#8242; splice site16,17,18. The expression of E1A variants changes according to the period of Adenoviral infection19,20.
We have shown previously that both Nek4 isoforms interact with splicing factors such as SRSF1 and hnRNPA1 and while isoform 2 changes minigene E1A alternative splicing, isoform 1 has no effect in that21. Because isoform 1 is the most abundant isoform and changes chemotherapy resistance and DNA damage response, we evaluate if it could change minigene E1A alternative splicing in a stress condition.
Minigene assay is a simple, low-cost and rapid method, since it only needs RNA extraction, cDNA synthesis, amplification and agarose gel analyses, and can be a useful tool to evaluate since a possible effect on alternative splicing by a protein of interests until the effect of different treatments on cellular alternative splicing pattern.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 pb DNA Ladder</td>
			<td>Invitrogen</td>
			<td>15628-050</td>
			<td></td>
		</tr>
		<tr>
			<td>6 wells plate</td>
			<td>Sarstedt</td>
			<td>833920</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>Sigma</td>
			<td>P4394</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC water</td>
			<td>ThermoFisher</td>
			<td>AM9920</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>11965118</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>ThermoFisher</td>
			<td>10297-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - FBS</td>
			<td>ThermoFisher</td>
			<td>12657029</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Leica</td>
			<td>DMIL LED FLUO</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel imaging acquisition system - ChemiDoc Gel Imagin System</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GFP - pEGFPC3</td>
			<td>Clontech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293 stable cells - HEK293 Flp-In</td>
			<td></td>
			<td></td>
			<td>Generated from Flp-In&#8482; T-REx&#8482; 293 - Invitrogen and described in ref 21</td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>ThermoFisher</td>
			<td>10687010</td>
			<td>Used for Flp-In cells maintenemant</td>
		</tr>
		<tr>
			<td>Image processing and analysis software - FIJI software</td>
			<td></td>
			<td></td>
			<td>ref. 32</td>
		</tr>
		<tr>
			<td>Lipid- based transfection reagent - jetOPTIMUS Polyplus Reagent</td>
			<td>Polyplus</td>
			<td>117-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo DT</td>
			<td>ThermoFisher</td>
			<td>18418020</td>
			<td></td>
		</tr>
		<tr>
			<td>Paclitxel</td>
			<td>Invitrogen</td>
			<td>P3456</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Reader/ UV absorbance</td>
			<td>Biotech</td>
			<td></td>
			<td>Epoch Biotek/ Take3 adapter</td>
		</tr>
		<tr>
			<td>pMTE1A plasmid</td>
			<td></td>
			<td></td>
			<td>Provided by Dr. Adrian Krainer</td>
		</tr>
		<tr>
			<td>pMTE1A F</td>
			<td>Invitrogen</td>
			<td>&#160;5&#8217; -ATTATCTGCCACGGAGGTGT-3</td>
			<td></td>
		</tr>
		<tr>
			<td>pMTE1A R</td>
			<td>Invitrogen</td>
			<td>5&#8217; -GGATAGCAGGCGCCATTTTA-3&#8217;</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>F5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse Transcriptase - M-MLV</td>
			<td>ThermoFisher</td>
			<td>28025013</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse transcriptase - Superscript IV</td>
			<td>ThermoFisher</td>
			<td>18090050</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribunuclease inhibitor RNAse OUT</td>
			<td>ThermoFisher</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA extraction phenol-chloroform based reagent - Trizol</td>
			<td>ThermoFisher</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>SybrSafe DNA gel stain</td>
			<td>ThermoFisher</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq Platinum</td>
			<td>Thermo</td>
			<td>10966026</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracyclin</td>
			<td>Sigma</td>
			<td>T3383</td>
			<td>Used for Flag empty or Nek4- Flag expression induction</td>
		</tr>
		<tr>
			<td>Thermocycler Bio-Rad</td>
			<td>Bio-Rad</td>
			<td>T100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4799</td>
			<td></td>
		</tr>
	</tbody>
</table>

using the e1a minigene tool to study mrna splicing changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

A 5&#180; splice sites&#160;assay using E1A minigene was performed to evaluate changes in splicing profile in cells after chemotherapy exposition. The role of Nek4 - isoform 1 in AS regulation in HEK293 stable cells after paclitaxel or cisplatin treatment was evaluated.
Adenoviral E1A region is responsible for the production of three main mRNAs from one RNA precursor because of the use of different splice donors. They share comm...

Watch this Scientific Journal Video about Using the E1A Minigene Tool to Study mRNA Splicing Changes at JoVE.com

Using the E1A Minigene Tool to Study mRNA Splicing Changes

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

The overall goal of this protocol is to provide detailed guidance to use the minigene E1A2 for evaluating global mRNA splicing changes. This is a quick, inexpensive, and simple tool for assessing the function of the target protein in alternative splicing regulation without the necessity of special regimes or laboratory conditions. Begin by culturing HEK 293 cells with stable expression of the gene of interest.Split the cells using 0.25%trypsin EDTA, then plate 300, 000 cells per well in six-well plates and incubate them for 24 hours at 37 degrees Celsius in 5%carbon dioxide. After 24 hours, check the confluence, and transfect HEK 293 stable cells only when 70 to 80%confluent. Carefully remove the cell culture medium with a pipette, then add two milliliters of complete DMEM medium without antibiotics and put the plate back in the incubator.Prepare a tube with the transfection buffer, then add two micrograms of pMTE1A DNA, vortex, and add two microliters of transfection reagent. Vortex again and incubate for 10 minutes at room temperature. Remove the plates from the incubator and carefully add the transfection mixture drop-wise.Six hours after the transfection, add tetracycline to HEK 293 stable cells for Nek4 expression induction. 24 hours after the transfection, verify cell morphology and transfection efficiency using a fluorescent microscope. Use a pipette to remove the cell culture medium, then add cell culture medium with the chemotherapeutics.Incubate the cells for another 24 hours. To collect RNA, discard the cell culture medium, and add 0.5 to 1 milliliter of RNA extraction reagent directly to the well. If wells are very confluent, use one milliliter of RNA extraction reagent to improve RNA quality.Homogenize the cells with a pipette and transfer them to a 1.5 milliliter centrifuge tube. Proceed immediately to the RNA extraction, or store the samples at minus 80 degrees Celsius. Thaw the samples in a fume hood and incubate for five minutes at room temperature.Add 0.1 to 0.2 milliliters of chloroform and agitate vigorously, then incubate for three minutes at room temperature. Centrifuge for 15 minutes at 12, 000 times G and four degrees Celsius, then collect the upper aqueous phase and transfer it to a new 1.5 milliliter centrifuge tube. Collect around 60%of the total volume, but do not collect the DNA or the organic phase.Add 0.25 to 0.5 milliliters of isopropanol and agitate the sample by inversion four times. Incubate for 10 minutes at room temperature, then centrifuge at 12, 000 times G for 10 minutes at four degrees Celsius and discard the supernatant. Wash the RNA pellet twice with ethanol, and centrifuge at 7, 500 times G for five minutes, then discard the ethanol.Remove excess ethanol by inverting the tube on a paper towel, then leave the tube open inside a fume hood to partially dry the pellet for five to 10 minutes. Re-suspend the RNA pellet in 15 microliters of DEPC-treated water. Quantify total RNA and verify RNA quality by measuring absorbance at 230, 260, and 280 nanometers.To verify total RNA quality, run a 1%agarose gel pretreated with 1.2%of a 2.5%sodium hypochlorite solution for 30 minutes. Perform cDNA synthesis using one to two micrograms of total RNA. Combine RNA, one microliter of oligo d-T, one microliter of DNTP, and nuclease-free water to 12 microliters.Incubate the reaction in the Thermo Cycler for five minutes at 65 degrees Celsius. Remove samples from the Thermo Cycler, and add four microliters of reverse transcriptase buffer, two microliters of DTT, and one microliter of ribonuclease inhibitor. Incubate at 37 degrees Celsius for two minutes.Add one microliter of thermo-stable reverse transcriptase, and incubate at 37 degrees Celsius for 50 minutes. Inactivate the enzyme at 70 degrees Celsius for 15 minutes. Perform PCR as described in the text manuscript, then load 20 to 25 microliters of the PCR product on a 3%agarose gel containing nucleic acid stain, and run at 100 volts for approximately one hour.Photograph the gel, avoiding any band saturation, and quantify the bands using an image processing software. The bands at 631, 493, and 156 base pairs correspond to the 13S, 12S, and 9S isoforms respectively. From the software's File menu, open the image file and convert it to gray scale.Adjust Brightness and Contrast, then remove outlier noise if necessary. Draw a rectangle around the first lane with the rectangle selection tool, and select it through the Analyze, Gels, and Select First Lane command, or by using the keyboard shortcut. Use the mouse to click and hold in the middle of the rectangle on the first lane, and drag it over to the next lane.Go to Analyze, Gels, and Select Next Lane. Repeat this step for all remaining lanes. After all the lanes are highlighted and numbered, go to Analyze, Gels, and Plot Lanes to draw a profile plot of each lane.Use the straight line selection tool to draw a line across the base of each peak, corresponding to each band, leaving out the background noise. After all the peaks from every lane have been closed off, select the wand tool and click inside each peak. Measurements should pop up in the results window for each highlighted peak.Consider only the 13S, 12S, and 9S peaks corresponding to 631, 493, and 156 base pairs bands. Copy intensity data for a spreadsheet editor, and sum the intensity from all three bands for each sample, then calculate the percentage for each isoform relative to the total. Plot the percentages of each isoform, or the differences in the percentage relative to the untreated samples.A plasmid containing the minigene from E1A was used to observe the effect on alternative splicing by evaluating the proportion of mRNA from each isoform produced. Basal expression of E1A isoforms variants depended on the cell line and time of E1A expression. The HEK 293 stable cell line, or HEK 293 recombinase containing site, showed a higher expression of 13S in comparison to HeLa cells, but showed similar levels of 13S and 12S isoforms after 48 hours of E1A expression.Cells exposed to cisplatin showed a shift in five prime S-S splicing selection favoring 12S expression. This effect was observed in HEK 293 stably expressing the Flag empty vector, as well as isoform one of Nek4. Changes in alternative splicing after paclitaxel treatment were very discreet, but the directions of the changes were opposite in Flag and Nek4 expressing cells.When performing this protocol, it's important to use untransfected and non-reverse transcriptase controls to avoid misinterpretation with endogenous E1A expression, mainly when using HEK 293 cells. After obtaining positive results, the alternative splicing of specific genes related to the chemotherapeutic response can be evaluated. When some effect is observed, this simple protocol can direct these studies for more consistent pathways, where the protein plays a role regulating alternative splicing in chemotherapy response, for example.

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