Name: a reporter based cellular assay for monitoring splicing efficiency
Uploaded: 2021-09-15T14:00:00
Description: Watch this Scientific Journal Video about A Reporter Based Cellular Assay for Monitoring Splicing Efficiency at JoVE.com

This work was supported by funds to S.S. from the National Institutes of Health (R21CA170786 and R01GM127464) and the American Cancer Society (the Institutional Research Grant 74-001-34-IRG) and to S.S. and W.M. from the Valley Research Partnership Program (P1-4009 and VRP77). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Reagents and buffers
NOTE: All sterilization using vacuum filters should be performed with 0.2 &#181;m polyethersulfone (PES) membrane in a biosafety cabinet.
<ol>
	<li>Prepare RNase-free water by adding 1.0 mL of diethylpyrocarbonate (DEPC) to 1.0 L of deionized water, mix for at least 1 hr at room temperature (RT), autoclave twice, and then cool to RT before use.</li>
	<li>Prepare Dulbecco&#39;s Modified Eagle Medium (DMEM) by mixing one packet of DMEM powder (13.4 g), 3.7 g of sodium b...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+atypical+5'+splice+sites+by+shifted+base-pairing+to+U1+snRNA.">Recognition of atypical 5' splice sites by shifted base-pairing to U1 snRNA.</a> Nature Structural Molecular Biology. 16 (2), 176-182 (2009).</li><li>Taladriz-Sender, A., Campbell, E., Burley, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splice-switching+small+molecules:+A+new+therapeutic+approach+to+modulate+gene+expression.">Splice-switching small molecules: A new therapeutic approach to modulate gene expression.</a> Methods. 167, 134-142 (2019).</li><li>Hamid, F. M., Makeyev, E. 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The assay can be adapted for splicing analysis in cell lines other than HeLa, however, factors affecting transfection efficiency, such as cell confluency and quantity of DNA may need to be optimized. The reporter to U1 construct ratio is another critical parameter that may need to be determined depending upon the expression levels observed in other cell types. The quality of extracted RNA is critical for splicing analysis; therefore, the use of RNase-free water and decontamination of surfaces with RNase inactivating agen...

Pre-mRNA splicing is an essential processing step that removes non-coding introns and precisely ligates coding exons to form mature mRNA. Recognition of consensus sequences at exon-intron junctions, referred to as 5&#8242;-splice site and 3&#8242;-splice site, by components of the splicing machinery initiates the splicing process. The U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5&#8242;-splice site by base pairing of the U1 snRNA to the pre-mRNA1. Genetically inherited mutations that alter 5&#8242;-splice site sequences are associated with many diseases2,3. It is predicted that the loss of basepairing of U1 snRNA with the mutant 5&#8242;-splice sites&#160;causes aberrant splicing, which can compromise translation of the affected transcript. A potential therapeutic approach to correct the splicing defects involves suppression of mutations by modified U1 snRNA carrying compensatory nucleotide changes in its 5&#8242;-region that basepairs with the 5&#8242;-splice site. Such modified U1 snRNAs, also referred to as exon specific U1 snRNAs, have been found to be effective in reversing splicing defects, resulting in increased protein expression from the rescued mRNA4,5,6,7,8.
Here, we describe the U1 snRNP complementation assay, a reporter-based cellular splicing assay that allows assessment of the effect of 5&#8242;-ss mutations on splicing of an exon and can also be used for the development of modified U1 snRNAs to enable the rescue of exon inclusion. We also provide protocols for monitoring of the spliced reporter transcripts by primer extension and RT-PCR, and for determining the expression of modified U1 snRNAs by primer extension and RT-qPCR.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent Grade Deionized Water</td>
			<td>ThermoFisher Scientific</td>
			<td>23-751628</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate (DEPC)</td>
			<td>Sigma-Aldrich</td>
			<td>D5758-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) powder packet</td>
			<td>Gibco</td>
			<td>12100-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>ThermoFisher Scientific</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), Australian Source, Heat Inactivated</td>
			<td>Omega Scientific</td>
			<td>FB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S)</td>
			<td>Sigma-Aldrich</td>
			<td>P4458-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide, Standard Solution 1.0N</td>
			<td>Sigma-Aldrich</td>
			<td>S2567-16A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid, Certified ACS Plus, 36.5 to 38.0%</td>
			<td>ThermoFisher Scientific</td>
			<td>A144-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable PES Bottle Top Filters</td>
			<td>ThermoFisher Scientific</td>
			<td>FB12566510</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA Disodium Salt Dihydrate</td>
			<td>Amresco</td>
			<td>0105-2.5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% Trypsin (10x), no phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Bioreagent</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Bioreagent</td>
			<td>BP366-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium Hydrogen Phosphate Heptahydrate</td>
			<td>Fisher Bioreagent</td>
			<td>BP332-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Dihydrogen Phosphate</td>
			<td>Fisher Bioreagent</td>
			<td>BP362-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection medium - Opti-MEM&#8482; I Reduced Serum Medium, no phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection Reagent - Lipofectamine&#8482; 2000</td>
			<td>ThermoFisher Scientific</td>
			<td>13778150</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol&#8482; Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform (Approx. 0.75% Ethanol as Preservative/Molecular Biology)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP1145-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, Absolute (200 Proof), Molecular Biology Grade, Fisher BioReagents</td>
			<td>ThermoFisher Scientific</td>
			<td>BP2818-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol, Molecular Biology Grade, Fisher BioReagents</td>
			<td>ThermoFisher Scientific</td>
			<td>BP2618-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen (5 mg/ml)</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct-zol RNA Miniprep Kit</td>
			<td>Zymo Research</td>
			<td>R2052</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP, [&#947;-32P]- 6000Ci/mmol 150mCi/ml Lead, 1 mCi</td>
			<td>PerkinElmer</td>
			<td>NEG035C001MC</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase</td>
			<td>New England Biolabs</td>
			<td>M0201L</td>
			<td></td>
		</tr>
		<tr>
			<td>Size exclusion beands - Sephadex&#174; G-25</td>
			<td>Sigma-Aldrich</td>
			<td>G2580-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Size exclusion mini columns</td>
			<td>USA Scientific</td>
			<td>1415-0600</td>
			<td></td>
		</tr>
		<tr>
			<td>pBR322 DNA-MspI Digest</td>
			<td>New England Biolabs</td>
			<td>N3032S</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Molecular Weight Marker, 10-100 nt</td>
			<td>Affymetrix</td>
			<td>76410 100 UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase inactivating reagents - RNaseZAP&#8482;</td>
			<td>Sigma-Aldrich</td>
			<td>R2020-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Mix (10 mM ea)</td>
			<td>ThermoFisher Scientific</td>
			<td>18427013</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseOUT&#8482; Recombinant Ribonuclease Inhibitor</td>
			<td>ThermoFisher Scientific</td>
			<td>10777019</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse Transcriptase - M-MLV Reverse Transcriptase</td>
			<td>ThermoFisher Scientific</td>
			<td>28025013</td>
			<td>used for primer extension</td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td>10342020</td>
			<td></td>
		</tr>
		<tr>
			<td>Random Hexamers (50 &#181;M)</td>
			<td>ThermoFisher Scientific</td>
			<td>N8080127</td>
			<td></td>
		</tr>
		<tr>
			<td>Real time PCR mix - SYBR&#8482; Select Master Mix</td>
			<td>ThermoFisher Scientific</td>
			<td>4472903</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript&#8482; III Reverse Transcriptase</td>
			<td>ThermoFisher Scientific</td>
			<td>18080093</td>
			<td>used for cDNA preparation</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>ThermoFisher Scientific</td>
			<td>18080093</td>
			<td></td>
		</tr>
		<tr>
			<td>5X First-Strand Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>18080093</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide (&#8805;99.5%)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP228-100</td>
			<td>Review Material Safety Data Sheets</td>
		</tr>
		<tr>
			<td>Bromophenol Blue sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>114405-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene Cyanol FF</td>
			<td>Sigma-Aldrich</td>
			<td>2650-17-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base (White Crystals or Crystalline Powder/Molecular Biology)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid (Crystalline/Electrophoresis)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP168-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide: Bis-Acrylamide 19:1 (40% Solution/Electrophoresis)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP1406-1</td>
			<td>Review Material Safety Data Sheets</td>
		</tr>
		<tr>
			<td>Urea (Colorless-to-White Crystals or Crystalline Powder/Mol. Biol.)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP169-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium peroxodisulphate (APS) &#8805;98%, Pro-Pure, Proteomics Grade</td>
			<td>VWR</td>
			<td>M133-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmacote</td>
			<td>Sigma-Aldrich</td>
			<td>SL2-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#39;,N&#39;-Tetramethylethylenediamine (TEMED) &#8805;99%, Ultrapure</td>
			<td>VWR</td>
			<td>0761-25ML</td>
			<td>Review Material Safety Data Sheets</td>
		</tr>
		<tr>
			<td>Adjustable Slab Gel Systems, Expedeon</td>
			<td>VWR</td>
			<td>ASG-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Gel Wrap&#8482; Glass Plate Sets, 16.5 x 14.5cm</td>
			<td>VWR</td>
			<td>NGP-125NR</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Gel Wrap&#8482; Glass Plate Sets, 16.5 x 22.0cm</td>
			<td>VWR</td>
			<td>NGP-200NR</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Gel Wrap&#8482; Glass Plate Sets, 16.5 x 38.7cm</td>
			<td>VWR</td>
			<td>NGP-400NR</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Storage Phosphor Screens</td>
			<td>Sigma-Aldrich</td>
			<td>GE28-9564</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon&#8482; FLA 7000 Biomolecular Imager</td>
			<td>GE Healthcare</td>
			<td>28-9610-73 AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter LS6500 Liquid Scintillation Counter</td>
			<td>GMI</td>
			<td>8043-30-1194</td>
			<td></td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 6 Flex Real-Time PCR Systems</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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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.

The splicing reporter Dup51, a three exon-two intron minigene, was derived from the human &#946;-globin gene and has been described previously (Figure 1A)11,12 . We created a mutant reporter, Dup51p, by introducing Usher syndrome associated 5&#180;-splice site mutations that occur in exon 3 of the protocadherin 15 (PCDH15) gene13. The 5&#180;-splice site sequence at the exon 2-intron 2 junction was ch...

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A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

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 ...

This protocol describes a cellular Assay for Monitoring Splicing Efficiency, using a adaptable managing reporter. This reporter Assay can be used to study the effects of disease Associated mutations in the Exxon or splice sites, and to design therapy, particular one particles, to improve the recognition of mutant to five point splice sites. The passage in Hela cells aspirate the spent medium and incubate the cells with three milliliters of 0.2 5%trips in containing one millimolar EDTA, at 37 degrees Celsius for three minutes.After incubation at seven milliliters of fresh DMEM and transfer the cell suspension to a 10 milliliter tube. Centrifuge the cells. Then resuspend the cell pellet in 10 milliliters of fresh DMEM, and plate the cells on a new tissue culture dish at 20%confluence, for transient transsfection's.Count the hela cells with a clean hemo cytometer slide and prepare a suspension with a density of 2.5 times 10 to the fifth cells per milliliter, seed one milliliter of the cell suspension into each well of a 12 well plate and incubate overnight at 37 degrees Celsius. The following day aspirate the spent medium and add 800 microliters of fresh DMEM with serum to the plate. Prepare the transfection mixes and Vortex them for 15 seconds.Then centrifuge the mixes and incubate them at room temperature for five minutes. After the incubation add all 200 microliters of the transfection mix into one well of the 12 well hela cell plate to achieve a final volume of one milliliter per well, and incubate the plate at 37 degrees Celsius for 48 hours. The prepared labeling reactions are incubating.Resuspend the 25 kilo Dalton molecular weight cutoff size exclusion beads by gentle vortexing for 10 seconds. Next transfer 600 microliters of the resuspended beads to a disposable mini column, placed in a 1.5 milliliter collection tube, and return the beads stock to four degree Celsius. Centrifuge the column and discard the flow through them.Then wash the beads twice by adding 300 microliters of RNA free water to the column. After the second wash, transfer the mini column to a new 1.5 milliliter centrifuge tube, and add the kinase reaction mix to the column. Centrifuge the column and collect the flow through containing the P32 labeled oligonucleotides.Add two micrograms of RNA extracted from the heli cells into 200 microliter micro centrifuge tubes and add RNA free water to make up the volume to 6.55 microliters. Next add 0.9 microliters a master mix one to each PCR tube containing the RNA samples, and incubate the tubes first at 65 degrees Celsius for five minutes, and then on ice for one minute. Next at 2.55 microliters, a master mix two, to each tube containing the RNA and master mix one, and incubate the tubes at room temperature for 10 minutes.After the incubation transfer the tubes to a thermal cycler and incubate first at 50 degrees Celsius for 60 minutes, and then at 70 degrees Celsius, for 15 minutes. After the incubation, add 10 microliters of 2X formamide RNA loading dye to each sample, and proceed to separate the fragments by UIA page. Percy DNA synthesis.Add two micrograms of RNA extracted from the transected hela cells, into 200 microliter microncentrifuge tubes and add RNAs free water to make up the volume to a low 11 microliters. Next, add two microliters, a master mix one to each sample and incubate first at 65 degree Celsius for five minutes, then on ice for one minute. Next, add seven microliters, a master mix two to each tube and incubate the tubes at room temperature for 10 minutes, followed by incubation at 50 degree Celsius for 60 minutes and 70 degrees Celsius for 15 minutes.Next, for PCR dilute the C DNA reaction to yield a concentration of 100 nanograms per microliter, and transfer one microliter of each CDNA sample to new PCR tubes. Add 10.5 microliters of the master mix to each tube containing CDNA, and perform the PCR. Using a thermocycler After the PCR is complete.Add 12.5 microliters of 2X formamide DNA loading dye to each tube. Heat the samples at 95 degree Celsius for five minutes, and proceed to perform a UIA page. Pipette five microliters of diluted CDNA into individual Wells of a 96-well qpcr plate triplicate.Next add 10 microliters of realtime PCR mix to each well, seal the plates with an optical film, and centrifuge to collect the reactions to the bottom of the Wells, then perform the qpcr while collecting the threshold quantification cycle values of target amplicon. Before loading the markers and samples, flush out the Wells with TBE buffer to remove the settled urea. Then load 10 microliters of each sample per well and run the gel at 300 to 500 volts for two to three hours, or until the xylene reaches the bottom.After electrophoresis remove the glass plates from the electrophoresis apparatus and carefully separate the two plates so that the gel lays flat on either glass surface, then transfer the gel onto a filter paper and cover it with a plastic wrap, using a gel dryer vacuum dry the gel at 80 degrees Celsius for 30 minutes, then place the dried gel in a phosphor imaging cassette for overnight incubation at room temperature. When expressed in hela cells, the slicing reporter dup 51 minnijean expresses the mature full length transcript in which Exxon two is spliced efficiently. The five prime splice site mutations and dup 51 P reduce Exxon two inclusion from 95 to 30%leading to the formation of a shorter transcript.Co-expression of dup 51 P with U15A SNRNA rescues Exxon two splicing, and restores the formation of the full length transcript. In contrast, co-expression with wild type U1 or U15G variant does not have the similar effect on dup 51P splicing. Detection of U15A variant transcripts, by primary extension uses the U1 seven to 26 oligo nucleotide, which is 20 nucleotides long and base pairs near the adenine at the fifth position of U1 SNRNA.In the presence of DATP alone, U1 seven through 26 allows incorporating two to three nucleotides for the endogenous wild type U1 SNRNA yielding a 22 or 23 nucleotide long product. For U15A and U15G variants extension terminates after adding a single adenosine producing a 21 nucleotide product. After normalization to U2 SNRNA expression, transfected U1 wild-type U1 5g and U15A variants were expressed at three to five folds over the endogenous SNRNA.The quality of the extracted RNA is critical for splicing analysis. Therefore, the use of RNA free water, and decontamination of services with RNAs inactivate agents is highly recommended. The report system described here can be applied to study the role of U1 SN RNAs in splicing regulations for reversing effect of flash price splicing mutations for gene silencing using modify U1SN RNAs.

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