Using the E1A Minigene Tool to Study mRNA Splicing Changes

Summary

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.

Abstract

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5´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.

Introduction

Splicing is among the most important steps in eukaryotic mRNA processing that occurs simultaneously to 5´mRNA capping and 3´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 splicing^2,3. 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´SS and 3´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 factors^6,7,8. 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 addressed^13,14. 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 events^13,14, however, as a preliminary assay, the minigene E1A¹⁵ is a very well established alternative splicing reporter system for the study of 5´SS selection in vivo. From only one gene, E1A, five mRNAs are produced by alternative splicing based on selection of three different 5′ splice sites and of one major or one minor 3′ splice site^16,17,18. The expression of E1A variants changes according to the period of Adenoviral infection^19,20.

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.

Protocol

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, HeLa^23,24,25,26, 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.

1. Plate cells considering vehicle, untransfected and GFP controls.
2. Culture HEK293 cells with stable expression of the gene of interest in Dulbecco'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 µg/mL of hygromycin B, on tissue culture-treated plates at 37 °C in 5% CO₂ and a humidified atmosphere.
3. Split cells using 0.25% trypsin-EDTA. Plate 3 x 10⁵ cells in 6-well plates and incubate them for 24 h at 37 °C in 5% CO₂.
   ​NOTE: For transfection, cells must be 70-80% confluent to decrease cell death after the transfection.

2. Cell transfection

NOTE: 1-2 µg 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 µg of pMTE1A DNA. For the transfection described here, a lipid-based transfection reagent was used

1. Check cell confluence 24 h after plating and transfect HEK293 stable cells only when 70-80% confluent.
2. 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.
3. Prepare a tube with the transfection buffer (200 µL/well), add 2 µg of pMTE1A DNA, vortex and then add 2 µL of transfection reagent. Vortex again and incubate for 10 min at room temperature.
4. Remove plates from the incubator and carefully add the transfection mixture dropwise.
5. To HEK293 stable cells, add tetracycline (0.5 µg/mL) for Nek4 expression induction 6 h after the transfection. Medium change is not necessary.
   ​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.
6. 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.
7. 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.

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.

1. Perform a dose-response curve before starting the assay to determine the minimal concentration to alternative splicing induction with no effect in cell viability.
2. Prepare a Paclitaxel stock solution at 5 mM concentration in ethanol. The final concentration is 1 µM. Keep at -20 °C. Use 0.02% ethanol as vehicle control.
3. 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 °C for 30 min. The final concentration is 30 µM. Prepare fresh or store at 2-10 °C for until one month.
   ​NOTE: All drugs must be protected from the light.

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.

1. Use RNA/DNase free tips and tubes. Perform total RNA extraction with phenol-chloroform reagent following manufacturer's recommendation.
2. 24 h after the transfection, verify cell morphology and transfection efficiency using a fluorescent microscope.
3. 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.
4. Incubate at 37 °C, 5% CO₂ for 18 - 24 h.
5. 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.
6. 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 °C or proceed immediately to the RNA extraction.
   ​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.

5. RNA extraction and cDNA synthesis

1. 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.
2. Incubate for 3 min at room temperature.
3. Centrifuge for 15 min at 12,000 x g and 4 °C.
4. 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.
5. Add 0.25 - 0.5 mL of isopropanol and agitate by inversion 4 times. Incubate for 10 min at room temperature.
6. Centrifuge at 12,000 x g for 10 min, at 4 °C and discard the supernatant.
7. 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.
8. 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.
9. Resuspend the RNA pellet in 15 µL of DEPC-treated water.
10. Quantify total RNA using absorbance at 230 nm, 260 nm, and 280 nm to verify RNA quality.
11. 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.
12. Perform cDNA synthesis using 1-2 µg of total RNA.
    1. Pipette RNA, 1 µL of oligo-dT (50 µM), 1 µL of dNTP (10 mM) and make up the volume to 12 µL with nuclease free water. Incubate in the thermocycler for 5 min at 65 °C.
    2. Remove samples from the thermocycler to cool down and prepare the reaction mixture: 4 µL of reverse transcriptase buffer, 2 µL of dithiothreitol (DTT), and 1 µL of ribonuclease inhibitor. Incubate at 37 °C for 2 min.
    3. Add 1 µL of thermo-stable reverse transcriptase. Incubate at 37 °C for 50 min and inactivate the enzyme at 70 °C for 15 min.
       ​NOTE: The cDNA can be stored at -20 °C for several weeks. Perform a non-reverse transcriptase (NRT) control for genomic contamination from putative intron-retention events discrimination.

6. pMTE1A minigene PCR

1. Perform PCR with the reaction composition (1.5 mM of MgCl₂, 0.3 mM of dNTP mix, 0.5 µM of each primer, 2.5 U of a hot-start Taq Polymerase and 150 ng of cDNA) with the following conditions:
   94 °C for 2 min, 29 cycles of: 94 °C for 1 min, 50 °C for 2 min, ​72 °C for 2 min, 72 °C for 10 min.
2. Load 20-25 µL of the PCR product in a 3% agarose gel containing nucleic acid stain and run at 100 V for approximately 1 h.

7. Analysis of the gel using an image processing and analysis software³²

1. After the run, photograph the gel (using a gel imaging acquisition system) avoiding any band saturation and quantify the bands using an image processing software.
2. For quantification consider the bands at ~631 bp, ~493 bp, and ~156 bp to correspond to the 13S, 12S and 9S isoforms, respectively.
3. From the software'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.
4. 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.
5. 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.
6. After all the lanes are highlighted and numbered, go to Analyze | Gels | Plot Lanes to draw a profile plot of each lane.
7. 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.
8. Sum the intensity from all the 3 bands for each sample and calculate the percentage for each isoform relative to the total.
9. 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%.

Results

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 comm...

Discussion

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 cell^13,14. 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...

Materials

Name	Company	Catalog Number	Comments
100 pb DNA Ladder	Invitrogen	15628-050
6 wells plate	Sarstedt	833920
Agarose	Sigma	A9539-250G
Cisplatin	Sigma	P4394
DEPC water	ThermoFisher	AM9920
DMEM	ThermoFisher	11965118
dNTP mix	ThermoFisher	10297-018
Fetal Bovine Serum - FBS	ThermoFisher	12657029
Fluorescent Microscope	Leica	DMIL LED FLUO
Gel imaging acquisition system - ChemiDoc Gel Imagin System	Bio-Rad
GFP - pEGFPC3	Clontech
HEK293 stable cells - HEK293 Flp-In			Generated from Flp-In™ T-REx™ 293 - Invitrogen and described in ref 21
Hygromycin B	ThermoFisher	10687010	Used for Flp-In cells maintenemant
Image processing and analysis software - FIJI software			ref. 32
Lipid- based transfection reagent - jetOPTIMUS Polyplus Reagent	Polyplus	117-07
Oligo DT	ThermoFisher	18418020
Paclitxel	Invitrogen	P3456
Plate Reader/ UV absorbance	Biotech		Epoch Biotek/ Take3 adapter
pMTE1A plasmid			Provided by Dr. Adrian Krainer
pMTE1A F	Invitrogen	5’ -ATTATCTGCCACGGAGGTGT-3
pMTE1A R	Invitrogen	5’ -GGATAGCAGGCGCCATTTTA-3’
Refrigerated centrifuge	Eppendorf	F5810R
Reverse Transcriptase - M-MLV	ThermoFisher	28025013
Reverse transcriptase - Superscript IV	ThermoFisher	18090050
Ribunuclease inhibitor RNAse OUT	ThermoFisher	10777-019
RNA extraction phenol-chloroform based reagent - Trizol	ThermoFisher	15596018
SybrSafe DNA gel stain	ThermoFisher	S33102
Taq Platinum	Thermo	10966026
Tetracyclin	Sigma	T3383	Used for Flag empty or Nek4- Flag expression induction
Thermocycler Bio-Rad	Bio-Rad	T100
Trypsin	Sigma	T4799