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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Cancer Research

Using the E1A Minigene Tool to Study mRNA Splicing Changes

Published: April 22nd, 2021

DOI:

10.3791/62181

1Faculdade de Ciências Farmacêuticas, 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.

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.

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....

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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-SY5.......

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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.......

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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.......

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We thank Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, through Grant Temático 2017/03489-1 to JK and fellowship to FLB 2018/05350-3) and the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (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. Patrícia Moriel, Prof. Dr. Wanda Pereira Almeida, Prof. Dr. Marcelo Lancellotti and Prof. Dr. Karina Kogo Cogo Müller to allow us to use their laboratory space and equipment.

....

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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

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