Name: Using the E1A Minigene Tool to Study mRNA Splicing Changes (Video) | JoVE Journal
Uploaded: 2021-04-22T13:30:00.000+00:00
Duration: 10 min 25 s
Description: Learn to use the E1A minigene tool for evaluating global mRNA splicing changes in HEK 293 cells. This protocol provides a rapid, inexpensive method to assess protein function in alternative splicing regulation.

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5 minutes

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-SY5Y29. The pattern of expression of minigene E1A isoforms under basal conditions varies between these cells and should be characterized for each condition. This protocol is not limited to stable cell lines. The most common approach in evaluation of candidate protein is by transient co-transfection of increasing its amount with fixed amount of the minigene. The same protocol is suitable for knockout cells.
<ol>
	<li>Plate cells considering vehicle, untransfected and GFP controls.</li>
	<li>Culture HEK293 cells with stable expression of the gene of interest in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% of Fetal Bovine Serum (FBS), 4.5 g/L Glucose, 4 mM L-Glutamine and maintain with 100 &#181;g/mL of hygromycin B, on tissue culture-treated plates at 37 &#176;C in 5% CO2 and a humidified atmosphere.</li>
	<li>Split cells using 0.25% trypsin-EDTA. Plate 3 x 105 cells in 6-well plates and incubate them for 24 h at 37 &#176;C in 5% CO2. 
	&#8203;NOTE: For transfection, cells must be 70-80% confluent to decrease cell death after the transfection.</li>
</ol>
2. Cell transfection
NOTE: 1-2 &#181;g of pMTE1A minigene plasmid was used here, however the DNA amount, as well as the time of expression, must be kept at minimal to avoid toxicity. For example, high toxicity was observed in HeLa cells after 30 h of transfection with 1 &#181;g of pMTE1A DNA. For the transfection described here, a lipid-based transfection reagent was used
<ol>
	<li>Check cell confluence 24 h after plating and transfect HEK293 stable cells only when 70-80% confluent.</li>
	<li>Remove the cell culture medium carefully with a pipette, instead of using a vacuum pump system. Then carefully add 2 mL of complete DMEM medium without antibiotics and put the plate back in the incubator.</li>
	<li>Prepare a tube with the transfection buffer (200 &#181;L/well), add 2 &#181;g of pMTE1A DNA, vortex and then add 2 &#181;L of transfection reagent. Vortex again and incubate for 10 min at room temperature.</li>
	<li>Remove plates from the incubator and carefully add the transfection mixture dropwise.</li>
	<li>To HEK293 stable cells, add tetracycline (0.5 &#181;g/mL) for Nek4 expression induction 6 h after the transfection. Medium change is not necessary. 
	&#8203;NOTE: Volumes/amounts described in this section are for one well. Prepare the mixture for all wells used in the experiment in the same tube.</li>
	<li>Prepare one well to transfect with EGFP, or another fluorophore expressing plasmid to estimate the transfection efficiency. Better results can be observed with transfection efficiency of at least 40%, but good performances with lower transfection efficiency were previously observed.</li>
	<li>When using co-transfection (interest protein and minigene) keep a well with non-transfected cells to avoid obtaining results from endogenous mRNA. In the case of E1A, HEK293 cells already express the E1A gene30.</li>
</ol>
3. Preparing the drugs
NOTE: The time and concentration of treatment were chosen based on literature results, which point out changes in alternative splicing for some genes.
<ol>
	<li>Perform a dose-response curve before starting the assay to determine the minimal concentration to alternative splicing induction with no effect in cell viability.</li>
	<li>Prepare a Paclitaxel stock solution at 5 mM concentration in ethanol. The final concentration is 1 &#181;M. Keep at -20 &#176;C. Use 0.02% ethanol as vehicle control.</li>
	<li>Prepare a cisplatin solution by diluting in 0.9% NaCl at around 0.5 mg/mL (1.66 mM). Protect from light, vortex and incubate in a thermal bath, 37 &#176;C for 30 min. The final concentration is 30 &#181;M. Prepare fresh or store at 2-10 &#176;C for until one month. 
	&#8203;NOTE: All drugs must be protected from the light.</li>
</ol>
4. Cell treatment and collection
NOTE: HEK293 stable cells were collected 48 h after the transfection and for this were treated 24 h after the transfection because the highest Nek4 expression level is achieved within 48 h. However, high levels of 13S isoform expression (until 90%) were observed at this time. To decrease the proportion of 13S isoform, try to treat and collect cells 30 h after transfection maximum.
<ol>
	<li>Use RNA/DNase free tips and tubes. Perform total RNA extraction with phenol-chloroform reagent following manufacturer&#39;s recommendation.</li>
	<li>24 h after the transfection, verify cell morphology and transfection efficiency using a fluorescent microscope.</li>
	<li>Remove the cell culture medium, preferentially using a pipette instead of a vacuum pump system. Then add cell culture medium with the chemotherapeutics in the previously described final concentration.</li>
	<li>Incubate at 37 &#176;C, 5% CO2 for 18 - 24 h.</li>
	<li>Collect RNA by discarding the cell culture medium in a container and adding 0.5 - 1 mL of RNA extraction reagent directly to the well. If wells are very confluent, use 1 mL of RNA extraction reagent to improve RNA quality.</li>
	<li>Homogenize with the pipette and transfer to a 1.5 mL centrifuge tube. At this point, the protocol can be paused by storing samples at -80 &#176;C or proceed immediately to the RNA extraction. 
	&#8203;WARNING: The phenol-based RNA extraction reagent is toxic and all procedures should be performed in a chemical fume hood and the residues disposed properly.</li>
</ol>
5. RNA extraction and cDNA synthesis
<ol>
	<li>In a fume hood thaw the samples and incubate for 5 min at room temperature. Add 0.1 -0.2 mL of chloroform and agitate vigorously.</li>
	<li>Incubate for 3 min at room temperature.</li>
	<li>Centrifuge for 15 min at 12,000 x g and 4 &#176;C.</li>
	<li>Collect the upper aqueous phase and transfer to a new 1.5 mL centrifuge tube. Collect around 60% of the total volume; however, do not collect the DNA or the organic (lower) phase.</li>
	<li>Add 0.25 - 0.5 mL of isopropanol and agitate by inversion 4 times. Incubate for 10 min at room temperature.</li>
	<li>Centrifuge at 12,000 x g for 10 min, at 4 &#176;C and discard the supernatant.</li>
	<li>Wash the RNA pellet twice with ethanol (75% in diethylpyrocarbonate (DEPC)-treated water). Centrifuge at 7,500 x g for 5 min and discard the ethanol.</li>
	<li>Remove excess ethanol by inverting the tube on a towel paper and then leave the tube open inside a fume hood to partially dry the pellet for 5 - 10 min.</li>
	<li>Resuspend the RNA pellet in 15 &#181;L of DEPC-treated water.</li>
	<li>Quantify total RNA using absorbance at 230 nm, 260 nm, and 280 nm to verify RNA quality.</li>
	<li>To verify total RNA quality, run a 1% agarose gel pre-treated with 1.2% (v/v) of a 2.5% sodium hypochlorite solution for 30 min31.</li>
	<li>Perform cDNA synthesis using 1-2 &#181;g of total RNA.
	<ol>
		<li>Pipette RNA, 1 &#181;L of oligo-dT (50 &#181;M), 1 &#181;L of dNTP (10 mM) and make up the volume to 12 &#181;L with nuclease free water. Incubate in the thermocycler for 5 min at 65 &#176;C.</li>
		<li>Remove samples from the thermocycler to cool down and prepare the reaction mixture: 4 &#181;L of reverse transcriptase buffer, 2 &#181;L of dithiothreitol (DTT), and 1 &#181;L of ribonuclease inhibitor. Incubate at 37 &#176;C for 2 min.</li>
		<li>Add 1 &#181;L of thermo-stable reverse transcriptase. Incubate at 37 &#176;C for 50 min and inactivate the enzyme at 70 &#176;C for 15 min. 
		&#8203;NOTE: The cDNA can be stored at -20 &#176;C for several weeks. Perform a non-reverse transcriptase (NRT) control for genomic contamination from putative intron-retention events discrimination.</li>
	</ol>
	</li>
</ol>
6. pMTE1A minigene PCR
<ol>
	<li>Perform PCR with the reaction composition (1.5 mM of MgCl2, 0.3 mM of dNTP mix, 0.5 &#181;M of each primer, 2.5 U of a hot-start Taq Polymerase and 150 ng of cDNA) with the following conditions: 
	94&#160;&#176;C for 2 min,&#160;29&#160;cycles of: 94 &#176;C for 1 min, 50 &#176;C for 2 min,&#160;&#8203;72 &#176;C for 2 min,&#160;72&#160;&#176;C for 10 min.</li>
	<li>Load 20-25 &#181;L of the PCR product in a 3% agarose gel containing nucleic acid stain and run at 100 V for approximately 1 h.</li>
</ol>
7. Analysis of the gel using an image processing and analysis software32
<ol>
	<li>After the run, photograph the gel (using a gel imaging acquisition system) avoiding any band saturation and quantify the bands using an&#160;image processing software.</li>
	<li>For quantification consider the bands at ~631 bp, ~493 bp, and ~156 bp to correspond to the 13S, 12S and 9S isoforms, respectively.</li>
	<li>From the software&#39;s File menu, open the image file obtained from the imaging acquisition system. Convert to greyscale, adjust brightness and contrast and remove outlier noise if necessary.</li>
	<li>Draw a rectangle around the first lane with the Rectangle Selection tool and select it through the Analyze | Gels | Select First Lane command, or by pressing the keyboard shortcut for it.</li>
	<li>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 | Select Next Lane, or press the available shortcut. Repeat this step to all remaining lanes.</li>
	<li>After all the lanes are highlighted and numbered, go to Analyze | Gels | Plot Lanes to draw a profile plot of each lane.</li>
	<li>With the Straight-line selection tool, 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, has been closed off, select the Wand tool and click inside each peak. For each peak that highlighted, measurements should pop up in the Results window that appears.</li>
	<li>Sum the intensity from all the 3 bands for each sample and calculate the percentage for each isoform relative to the total.</li>
	<li>Plot the percentages of each isoform or the differences in the percentage relative to untreated samples. 
	NOTE: Ensure that the sum of three E1A variants must be equal to 100%.</li>
</ol>

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<li>Bai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+3%27+splice+site+choice+in+vivo+by+ASF%2FSF2+and+hnRNP+A1%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Control of 3' splice site choice in vivo by ASF/SF2 and hnRNP A1.</a> Nucleic Acids Research. 27 (4), 1126-1134 (1999).</li>
<li>Cote, G. J., Nguyen, N., Lips, C. J. M., Berget, S. M., Gagel, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Validation+of+an+in+vitro+RNA+processing+system+for+CT%2FCGRP+precursor+mRNA%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Validation of an in vitro RNA processing system for CT/CGRP precursor mRNA.</a> Nucleic Acids Research. 19 (13), 3601-3606 (1991).</li>
</ol>

The authors declare that they have no competing financial interests.

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><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&amp;trade; T-REx&amp;trade; 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>&amp;nbsp;5&amp;rsquo; -ATTATCTGCCACGGAGGTGT-3</td><td></td></tr><tr><td>pMTE1A R</td><td>Invitrogen</td><td>5&amp;rsquo; -GGATAGCAGGCGCCATTTTA-3&amp;rsquo;</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

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´ splice sites 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 common 5' and ...

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 ...

using-the-e1a-minigene-tool-to-study-mrna-splicing-changes

Research

JoVE Journal

Cancer Research

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and α/β C-termini, can be quantified.

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

This protocol describes a minigene reporter assay to monitor the impact of 5´-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (µTGGE) as a simple, quick, and portable method of detecting RNA editing.

A Nonsequencing Approach for the Rapid Detection of RNA Editing

Learn to use the E1A minigene tool for evaluating global mRNA splicing changes in HEK 293 cells. This protocol provides a rapid, inexpensive method to assess protein function in alternative splicing regulation.

Using the E1A Minigene Tool to Study mRNA Splicing Changes

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