Name: using the e1a minigene tool to study mrna splicing changes
Uploaded: 2021-04-22T13:30:00.000+00:00
Duration: 10 min 25 s
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-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>

<ol>
	<li>Ule, J., Blencowe, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+Splicing+Regulatory+Networks:+Functions,+Mechanisms,+and+Evolution.">Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution.</a> Molecular Cell. 76 (2), 329-345 (2019).</li><li>Pan, Q., Shai, O., Lee, L. J., Frey, B. J., Blencowe, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+surveying+of+alternative+splicing+complexity+in+the+human+transcriptome+by+high-throughput+sequencing.">Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.</a> Nature Genetics. 40 (12), 1413-1415 (2008).</li><li>Nilsen, T. W., Graveley, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+the+eukaryotic+proteome+by+alternative+splicing.">Expansion of the eukaryotic proteome by alternative splicing.</a> Nature. 463 (7280), 457-463 (2010).</li><li>Stamm, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signals+and+their+transduction+pathways+regulating+alternative+splicing:+a+new+dimension+of+the+human+genome.">Signals and their transduction pathways regulating alternative splicing: a new dimension of the human genome.</a> Human Molecular Genetics. 11 (20), 2409-2416 (2002).</li><li>Kornblihtt, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing:+a+pivotal+step+between+eukaryotic+transcription+and+translation.">Alternative splicing: a pivotal step between eukaryotic transcription and translation.</a> Nature Reviews Molecular Cell Biology. 14 (3), 153-165 (2013).</li><li>Zhong, X. -. Y., Ding, J. -. H., Adams, J. A., Ghosh, G., Fu, X. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+SR+protein+phosphorylation+and+alternative+splicing+by+modulating+kinetic+interactions+of+SRPK1+with+molecular+chaperones.">Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of SRPK1 with molecular chaperones.</a> Genes &amp; Development. 23 (4), 482-495 (2009).</li><li>Misteli, T., C&#225;ceres, J. F., Clement, J. Q., Krainer, A. R., Wilkinson, M. F., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serine+Phosphorylation+of+SR+Proteins+Is+Required+for+Their+Recruitment+to+Sites+of+Transcription+In+Vivo.">Serine Phosphorylation of SR Proteins Is Required for Their Recruitment to Sites of Transcription In Vivo.</a> Journal of Cell Biology. 143 (2), 297-307 (1998).</li><li>Kanopka, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+adenovirus+alternative+RNA+splicing+by+dephosphorylation+of+SR+proteins.">Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins.</a> Nature. 393 (6681), 185-187 (1998).</li><li>Anufrieva, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapy-induced+stress+response+is+associated+with+downregulation+of+pre-mRNA+splicing+in+cancer+cells.">Therapy-induced stress response is associated with downregulation of pre-mRNA splicing in cancer cells.</a> Genome Medicine. 10 (1), 49 (2018).</li><li>Shultz, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SRSF1+Regulates+the+Alternative+Splicing+of+Caspase+9+Via+A+Novel+Intronic+Splicing+Enhancer+Affecting+the+Chemotherapeutic+Sensitivity+of+Non-Small+Cell+Lung+Cancer+Cells.">SRSF1 Regulates the Alternative Splicing of Caspase 9 Via A Novel Intronic Splicing Enhancer Affecting the Chemotherapeutic Sensitivity of Non-Small Cell Lung Cancer Cells.</a> Molecular Cancer Research. 9 (7), 889-900 (2011).</li><li>Gabriel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+splicing+factor+SRSF4+in+cisplatin-induced+modifications+of+pre-mRNA+splicing+and+apoptosis.">Role of the splicing factor SRSF4 in cisplatin-induced modifications of pre-mRNA splicing and apoptosis.</a> BMC Cancer. 15, (2015).</li><li>Lee, S. C. -. W., Abdel-Wahab, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+targeting+of+splicing+in+cancer.">Therapeutic targeting of splicing in cancer.</a> Nature Medicine. 22 (9), 976-986 (2016).</li><li>Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+minigene+systems+to+dissect+alternative+splicing+elements.">Use of minigene systems to dissect alternative splicing elements.</a> Methods. 37 (4), 331-340 (2005).</li><li>Stoss, O., Stoilov, P., Hartmann, A. M., Nayler, O., Stamm, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+minigene+approach+to+analyze+tissue-specific+splicing.">The in vivo minigene approach to analyze tissue-specific splicing.</a> Brain Research Protocols. 4 (3), 383-394 (1999).</li><li>Zerler, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+E1A+coding+sequences+that+enable+ras+and+pmt+oncogenes+to+transform+cultured+primary+cells.">Adenovirus E1A coding sequences that enable ras and pmt oncogenes to transform cultured primary cells.</a> Molecular and Cellular Biology. 6 (3), 887-899 (1986).</li><li>Gattoni, R., Schmitt, P., Stevenin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+splicing+of+adenovirus+E1A+transcripts:+characterization+of+novel+reactions+and+of+multiple+branch+points+abnormally+far+from+the+3'+splice+site.">In vitro splicing of adenovirus E1A transcripts: characterization of novel reactions and of multiple branch points abnormally far from the 3' splice site.</a> Nucleic Acids Research. 16 (6), 2389-2409 (1988).</li><li>Stephens, C., Harlow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+splicing+yields+novel+adenovirus+5+E1A+mRNAs+that+encode+30+kd+and+35+kd+proteins.">Differential splicing yields novel adenovirus 5 E1A mRNAs that encode 30 kd and 35 kd proteins.</a> The EMBO journal. 6 (7), 2027-2035 (1987).</li><li>Ulfendahl, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+adenovirus-2+E1A+mRNA+encoding+a+protein+with+transcription+activation+properties.">A novel adenovirus-2 E1A mRNA encoding a protein with transcription activation properties.</a> The EMBO journal. 6 (7), 2037-2044 (1987).</li><li>Berk, A. J., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+adenovirus+2+early+mRNAs.">Structure of the adenovirus 2 early mRNAs.</a> Cell. 14 (3), 695-711 (1978).</li><li>Svensson, C., Pettersson, U., Akusj&#228;rvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+of+adenovirus+2+early+region+1A+mRNAs+is+non-sequential.">Splicing of adenovirus 2 early region 1A mRNAs is non-sequential.</a> Journal of Molecular Biology. 165 (3), 475-495 (1983).</li><li>Basei, F. L., Meirelles, G. V., Righetto, G. L., dos Santos Migueleti, D. L., Smetana, J. H. C., Kobarg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+interaction+partners+for+Nek4.1+and+Nek4.2+isoforms:+from+the+DNA+damage+response+to+RNA+splicing.">New interaction partners for Nek4.1 and Nek4.2 isoforms: from the DNA damage response to RNA splicing.</a> Proteome Science. 13 (1), 11 (2015).</li><li>Zhou, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Akt-SRPK-SR+Axis+Constitutes+a+Major+Pathway+in+Transducing+EGF+Signaling+to+Regulate+Alternative+Splicing+in+the+Nucleus.">The Akt-SRPK-SR Axis Constitutes a Major Pathway in Transducing EGF Signaling to Regulate Alternative Splicing in the Nucleus.</a> Molecular Cell. 47 (3), 422-433 (2012).</li><li>Caceres, J., Stamm, S., Helfman, D., Krainer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+alternative+splicing+in+vivo+by+overexpression+of+antagonistic+splicing+factors.">Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors.</a> Science. 265 (5179), 1706-1709 (1994).</li><li>Zhong, X. -. Y., Ding, J. -. H., Adams, J. A., Ghosh, G., Fu, X. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+SR+protein+phosphorylation+and+alternative+splicing+by+modulating+kinetic+interactions+of+SRPK1+with+molecular+chaperones.">Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of SRPK1 with molecular chaperones.</a> Genes &amp; Development. 23 (4), 482-495 (2009).</li><li>Naro, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+centrosomal+kinase+NEK2+is+a+novel+splicing+factor+kinase+involved+in+cell+survival.">The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival.</a> Nucleic Acids Research. 42 (5), 3218-3227 (2014).</li><li>Lu, C. -. C., Chen, T. -. H., Wu, J. -. R., Chen, H. -. H., Yu, H. -. Y., Tarn, W. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+and+Molecular+Characterization+of+the+Splicing+Factor+RBM4.">Phylogenetic and Molecular Characterization of the Splicing Factor RBM4.</a> PLoS ONE. 8 (3), 59092 (2013).</li><li>Jarn&#230;ss, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+Factor+Arginine/Serine-rich+17A+(SFRS17A)+Is+an+A-kinase+Anchoring+Protein+That+Targets+Protein+Kinase+A+to+Splicing+Factor+Compartments.">Splicing Factor Arginine/Serine-rich 17A (SFRS17A) Is an A-kinase Anchoring Protein That Targets Protein Kinase A to Splicing Factor Compartments.</a> Journal of Biological Chemistry. 284 (50), 35154-35164 (2009).</li><li>Bressan, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+association+of+human+Ki-1/57+with+pre-mRNA+splicing+events.">Functional association of human Ki-1/57 with pre-mRNA splicing events.</a> FEBS Journal. 276 (14), 3770-3783 (2009).</li><li>Vivarelli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraquat+Modulates+Alternative+Pre-mRNA+Splicing+by+Modifying+the+Intracellular+Distribution+of+SRPK2.">Paraquat Modulates Alternative Pre-mRNA Splicing by Modifying the Intracellular Distribution of SRPK2.</a> PLoS ONE. 8 (4), 61980 (2013).</li><li>Russell, W. C., Graham, F. L., Smiley, J., Nairn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+a+Human+Cell+Line+Transformed+by+DNA+from+Human+Adenovirus+Type+5.">Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5.</a> Journal of General Virology. 36 (1), 59-72 (1977).</li><li>Aranda, P. S., LaJoie, D. M., Jorcyk, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleach+gel:+A+simple+agarose+gel+for+analyzing+RNA+quality.">Bleach gel: A simple agarose gel for analyzing RNA quality.</a> Electrophoresis. 33 (2), 366-369 (2012).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Bai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+3'+splice+site+choice+in+vivo+by+ASF/SF2+and+hnRNP+A1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+an+in+vitro+RNA+processing+system+for+CT/CGRP+precursor+mRNA.">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

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

4.7K Views.  Universidade Estadual de Campinas. 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.

Video: Using the E1A Minigene Tool to Study mRNA Splicing Changes

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

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

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 &#945;/&#946; C-termini, can be quantified.

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

Genetics

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

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is 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 several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

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

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.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

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

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

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

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

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

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.

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Biochemistry

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

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

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.

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-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.

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Biology

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

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.

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

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.

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

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

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

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

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

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

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

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data