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). T....

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 charac....

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