A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

Summary

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

Abstract

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

Introduction

Precursor mRNA splicing is an important process for gene expression regulation in eukaryotes¹. The removal of introns and the union of exons in mature RNA is catalyzed by the spliceosome, a 2 megadalton ribonucleoprotein complex composed of 5 snRNAs (U1, U2, U4, U5, and U6) along with more than 100 proteins^2,3. The splicing reaction occurs co-transcriptionally, and the spliceosome is assembled at each new intron guided by the recognition of conserved splice sites at exon-intron boundaries and within the intron⁴. Different introns might have different splicing rat....

Protocol

An overview of the protocol described here is depicted in Figure 1.

1. Plasmid construction

1. pCAGGS-Cre: This plasmid was provided by Dr. E. Makeyev²¹.
2. pRD-miR-17-92:
   1. Amplify pre-miR-17-92 by PCR using 0.5 µM of each specific primer (Table of Materials), 150 ng of cDNA, 1 mM dNTPs, 1x Taq PCR buffer, and 5 U of high-fidelity Taq DNA polymerase. Perform a no-template control PCR reaction (use water instead of cDNA) to check for DNA contamination.
   2. Ligate the PCR product into pRD-RIPE (kindly provide....

Results

Our initial hypothesis was that HuR could facilitate intronic miRNA biogenesis by binding to its pre-miRNA sequence. Thus, the connection of HuR expression and miR-17-92 cluster biogenesis could point to a new mechanism governing the maturation of these miRNAs. Overexpression of HuR upon transfection of pFLAG-HuR was confirmed in three different cell lines: HeLa, BCPAP, and HEK-293T (Figure 2). As controls, untransfected cells and cells transfected with empty pFLAG vectors were used.......

Discussion

Pre-mRNA splicing is an important process for gene expression regulation, and its control can trigger strong effects on cell phenotypic modifications^22,23. More than 70% of miRNAs are transcribed from introns in humans, and we hypothesized that their processing and maturation could be facilitated by splicing regulatory proteins^24,25. We developed a method to analyze intronic miRNA processing and function .......

Materials

Name	Company	Catalog Number	Comments
Recombinant DNA
pCAGGS-Cre (Cre- encoding plasmid)			A kind gift from E. Makeyev from Khandelia et al., 2011
pFLAG-HuR			Generated during this work
pmiRGLO-RAP-IB			Generated during this work
pmiRGLO-scrambled			Generated during this work
pRD-miR-17-92			Generated during this work
pRD-RIPE-donor			A kind gift from E. Makeyev from Khandelia et al., 2011
pTK-Renilla	Promega	E2241
Antibodies
anti-B-actin	Sigma Aldrich	A5316
anti-HuR	Cell Signaling	mAb 12582
IRDye 680CW Goat anti-mouse IgG	Li-Cor Biosciences	926-68070
IRDye 800CW Goat anti-rabbit IgG	Li-Cor Biosciences	929-70020
Experimental Models: Cell Lines
HeLa-Cre			A kind gift from E. Makeyev from Khandelia et al., 2011
HeLa-Cre miR17-92			Generated during this work
HeLa-Cre miR17-92-HuR			Generated during this work
HeLa-Cre miR17-92-HuR-luc			Generated during this work
HeLa-Cre miR17-92-luc			Generated during this work
HeLa-Cre miR17-92-scrambled			Generated during this work
Chemicals and Peptides
DMEM/high-glucose	Thermo Fisher Scientific	12800-017
Doxycycline	BioBasic	MB719150
Dual-Glo Luciferase Assay System	Promega	E2940
EcoRI	Thermo Fisher Scientific	ER0271
EcoRV	Thermo Fisher Scientific	ER0301
Geneticin	Thermo Fisher Scientific	E859-EG
L-glutamine	Life Technologies
Opti-MEM I	Life Technologies	31985-070
pFLAG-CMV™-3 Expression Vector	Sigma Aldrich	E6783
pGEM-T	Promega	A3600
Platinum Taq DNA polymerase	Thermo Fisher Scientific	10966-030
pmiR-GLO	Promega	E1330
Puromycin	Sigma Aldrich	P8833
RNAse OUT	Thermo Fisher Scientific	752899
SuperScript IV kit	Thermo Fisher Scientific	18091050
Trizol-LS reagent	Thermo Fisher	10296-028
trypsin/EDTA 10X	Life Technologies	15400-054
XbaI	Thermo Fisher Scientific	10131035
XhoI	Promega	R616A
Oligonucleotides
forward RAP-1B pmiRGLO	Exxtend	TCGAGTAGCGGCCGCTAGTAAG CTACTATATCAGTTTGCACAT
reverse RAP-1B pmiRGLO	Exxtend	CTAGATGTGCAAACTGATATAGT AGCTTACTAGCGGCCGCTAC
forward scrambled pmiRGLO	Exxtend	TCGAGTAGCGGCCGCTAGTAA GCTACTATATCAGGGGTAAAAT
reverse scrambled pmiRGLO	Exxtend	CTAGATTTTACCCCTGATATAGT AGCTTACTAGCGGCCGCTAC
forward HuR pFLAG	Exxtend	GCCGCGAATTCAATGTCTAAT GGTTATGAAGAC
reverse HuR pFLAG	Exxtend	GCGCTGATATCGTTATTTGTG GGACTTGTTGG
forward pre-miR-1792 pRD-RIPE	Exxtend	ATCCTCGAGAATTCCCATTAG GGATTATGCTGAG
reverse pre-miR-1792 pRD-RIPE	Exxtend	ACTAAGCTTGATATCATCTTG TACATTTAACAGTG
forward snRNA U6 (RNU6B)	Exxtend	CTCGCTTCGGCAGCACATATAC
reverse snRNA U6 (RNU6B)	Exxtend	GGAACGCTTCACGAATTTGCGTG
forward B-Actin qPCR	Exxtend	ACCTTCTACAATGAGCTGCG
reverse B-Actin qPCR	Exxtend	CCTGGATAGCAACGTACATGG
forward HuR qPCR	Exxtend	ATCCTCTGGCAGATGTTTGG
reverse HuR qPCR	Exxtend	CATCGCGGCTTCTTCATAGT
forward pre-miR-1792 qPCR	Exxtend	GTGCTCGAGACGAATTCGTCA GAATAATGTCAAAGTG
reverse pre-miR-1792 qPCR	Exxtend	TCCAAGCTTAAGATATCCCAAAC TCAACAGGCCG
Software and Algorithms
Prism 8 for Mac OS X	Graphpad	https://www.graphpad.com
ImageJ	National Institutes of Health	http://imagej.nih.gov/ij