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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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....

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-tem.......

Representative 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 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 .......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Recombinant DNA
pCAGGS-Cre (Cre- encoding plasmid)A kind gift from E. Makeyev from Khandelia et al., 2011
pFLAG-HuRGenerated during this work
pmiRGLO-RAP-IBGenerated during this work
pmiRGLO-scrambledGenerated during this work
pRD-miR-17-92Generated during this work
pRD-RIPE-donorA kind gift from E. Makeyev from Khandelia et al., 2011
pTK-RenillaPromegaE2241
Antibodies
anti-B-actinSigma AldrichA5316
anti-HuRCell SignalingmAb 12582
IRDye 680CW Goat anti-mouse IgGLi-Cor Biosciences926-68070
IRDye 800CW Goat anti-rabbit IgGLi-Cor Biosciences929-70020
Experimental Models: Cell Lines
HeLa-CreA kind gift from E. Makeyev from Khandelia et al., 2011
HeLa-Cre miR17-92Generated during this work
HeLa-Cre miR17-92-HuRGenerated during this work
HeLa-Cre miR17-92-HuR-lucGenerated during this work
HeLa-Cre miR17-92-lucGenerated during this work
HeLa-Cre miR17-92-scrambledGenerated during this work
Chemicals and Peptides
DMEM/high-glucoseThermo Fisher Scientific12800-017
DoxycyclineBioBasicMB719150
Dual-Glo Luciferase Assay SystemPromegaE2940
EcoRIThermo Fisher ScientificER0271
EcoRVThermo Fisher ScientificER0301
GeneticinThermo Fisher ScientificE859-EG
L-glutamineLife Technologies
Opti-MEM ILife Technologies31985-070
pFLAG-CMV™-3 Expression VectorSigma AldrichE6783
pGEM-TPromegaA3600
Platinum Taq DNA polymeraseThermo Fisher Scientific10966-030
pmiR-GLOPromegaE1330
PuromycinSigma AldrichP8833
RNAse OUTThermo Fisher Scientific752899
SuperScript IV kitThermo Fisher Scientific18091050
Trizol-LS reagentThermo Fisher10296-028
trypsin/EDTA 10XLife Technologies15400-054
XbaIThermo Fisher Scientific10131035
XhoIPromegaR616A
Oligonucleotides
forward RAP-1B pmiRGLOExxtendTCGAGTAGCGGCCGCTAGTAAG
CTACTATATCAGTTTGCACAT
reverse RAP-1B pmiRGLOExxtendCTAGATGTGCAAACTGATATAGT
AGCTTACTAGCGGCCGCTAC
forward scrambled pmiRGLOExxtendTCGAGTAGCGGCCGCTAGTAA
GCTACTATATCAGGGGTAAAAT
reverse scrambled pmiRGLOExxtendCTAGATTTTACCCCTGATATAGT
AGCTTACTAGCGGCCGCTAC
forward HuR pFLAGExxtendGCCGCGAATTCAATGTCTAAT
GGTTATGAAGAC
reverse HuR pFLAGExxtendGCGCTGATATCGTTATTTGTG
GGACTTGTTGG
forward pre-miR-1792 pRD-RIPEExxtendATCCTCGAGAATTCCCATTAG
GGATTATGCTGAG
reverse pre-miR-1792 pRD-RIPEExxtendACTAAGCTTGATATCATCTTG
TACATTTAACAGTG
forward snRNA U6 (RNU6B)ExxtendCTCGCTTCGGCAGCACATATAC
reverse snRNA U6 (RNU6B)ExxtendGGAACGCTTCACGAATTTGCGTG
forward B-Actin qPCRExxtendACCTTCTACAATGAGCTGCG
reverse B-Actin qPCRExxtendCCTGGATAGCAACGTACATGG
forward HuR qPCRExxtendATCCTCTGGCAGATGTTTGG
reverse HuR qPCRExxtendCATCGCGGCTTCTTCATAGT
forward pre-miR-1792 qPCRExxtendGTGCTCGAGACGAATTCGTCA
GAATAATGTCAAAGTG
reverse pre-miR-1792 qPCRExxtendTCCAAGCTTAAGATATCCCAAAC
TCAACAGGCCG
Software and Algorithms
Prism 8 for Mac OS XGraphpadhttps://www.graphpad.com
ImageJNational Institutes of Healthhttp://imagej.nih.gov/ij

References

  1. Wilkinson, M. E., Charenton, C., Nagai, K. RNA splicing by the spliceosome. Annual Review of Biochemistry. 89, 359-388 (2020).
  2. Will, C. L., Luhrmann, R. Spliceosome structure and function. Cold Spring Harbor Perspectives in Biology. 3 (7),....

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