A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

Summary

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

Abstract

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′ 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′ region that basepairs with the 5′-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′ splice-sites. Insertion of cis-acting 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′ 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.

Introduction

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′-splice site and 3′-splice site, by components of the splicing machinery initiates the splicing process. The U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5′-splice site by base pairing of the U1 snRNA to the pre-mRNA¹. Genetically inherited mutations that alter 5′-splice site sequences are associated with many diseases

Protocol

1. Reagents and buffers

NOTE: All sterilization using vacuum filters should be performed with 0.2 µm polyethersulfone (PES) membrane in a biosafety cabinet.

1. 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.
2. Prepare Dulbecco's Modified Eagle Medium (DMEM) by mixing one packet of DMEM powder (13.4 g), 3.7 g of sodium bicarbonate, 100 mL of fetal bovine serum (FBS), penicillin, and streptomycin to ~800 mL of sterile deionized water. The final concentration of penicillin and s....

Results

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

Discussion

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.......

Materials

List of materials used in this article

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