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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.
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.
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 proteins2,3. 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....
An overview of the protocol described here is depicted in Figure 1.
1. Plasmid construction
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.......
Pre-mRNA splicing is an important process for gene expression regulation, and its control can trigger strong effects on cell phenotypic modifications22,23. 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 proteins24,25. We developed a method to analyze intronic miRNA processing and function .......
The authors are grateful to E. Makeyev (Nanyang Technological University, Singapore) for the HeLa-Cre cells and pRD-RIPE and pCAGGS-Cre plasmids. We thank Edna Kimura, Carolina Purcell Goes, Gisela Ramos, Lucia Rossetti Lopes, and Anselmo Moriscot for their support.
....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 |
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