The Ago2-miRNA-co-IP Assay to Study TGF- β1 Mediated Recruitment of miRNA to the RISC in CFBE Cells

Published: July 31, 2020

doi:

Nilay Mitash², Joshua E. Donovan³, Agnieszka Swiatecka-Urban³

¹Department of Pediatrics, UPMC Children’s Hospital of Pittsburgh,University of Pittsburgh School of Medicine, ²Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, 200 Lothrop Street,University of Pittsburgh, Pittsburgh, PA, 15261, ³Department of Pediatric Nephrology,University of Virginia, Charlottesville, VA, 22908

Summary

Here, we describe the Ago2-miRNA-co-IP assay designed to quantify an active pool of specific miRNAs induced by TGF-β1 in human bronchial epithelial CFBE41o- cells. This assay provides functional information on the recruitment of miRNA to the RNA induced silencing complex, quantified by qRT-PCR using specific miRNA primers and TaqMan hydrolysis probes.

Abstract

Micro(mi)RNAs are short, non-coding RNAs that mediate the RNA interference (RNAi) by post-transcriptional mechanisms. Specific miRNAs are recruited to the cytoplasmic RNA induced silencing complex (RISC). Argonaute2 (Ago2), an essential component of RISC, facilitates binding of miRNA to the target-site on mRNA, followed by cleaving the miRNA-mRNA duplex with its endonuclease activity. RNAi is mediated by a specific pool of miRNAs recruited to RISC, and thus is referred to as the functional pool. The cellular levels of many miRNAs are affected by the cytokine Transforming Growth Factor-β1 (TGF-β1). However, little is known about whether the TGF-β1 affects the functional pools of these miRNAs. The Ago2-miRNA-co-IP assay, discussed in this manuscript, is designed to examine effects of TGF-β1 on the recruitment of miRNAs to RISC and it helps to determine whether changes in the cellular miRNA levels correlate with changes in the RISC-associated, functional pools. The general principles of the assay are as follows. Cultured cells treated with TGF-β1 or vehicle control are lysed and the endogenous Ago2 is immunoprecipitated with immobilized anti-Ago2 antibody, and the active miRNAs complexed with Ago2 are isolated with a RISC immunoprecipitation (RIP) assay kit. The miRNAs are identified with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) using miRNA-specific stem-looped primers during reverse transcription, followed by PCR using miRNA-specific forward and reverse primers, and TaqMan hydrolysis probes.

Introduction

Transforming Growth Factor-β1 (TGF-β1) is a multifunctional cytokine that can change the expression of many micro(mi)RNAs¹^,²^,³. The total cellular level of a particular miRNA does not correlate with its inhibitory potential because only a specific fraction of the miRNA is incorporated into RNA induced silencing complex (RISC) to perform RNA interference (RNAi)³. Only up to 10% of each miRNA is RISC-associated and participates in RNAi⁴^,⁵. Next, the RNAi process involves binding of the RISC-associated miRNA to the target mRNA recognition sequence(s)⁶. The RISC association is influenced by the availability of the target mRNA and the miRNA complementarity to the binding site, usually present at 3’ untranslated region (UTR) of the mRNA⁴. The Argonaute2-miRNA-co-immunoprecipitation (Ago2-miRNA-co-IP) assay, described in this manuscript, is designed to examine the effect of TGF-β1 on the recruitment of specific miRNAs to RISC by detecting differences in the RISC-associated miRNAs after TGF-β1 treatment, compared to the vehicle control. Examining the RISC-associated functional pool of a specific miRNA is much more informative about the miRNA effects than examining the total cellular level of the miRNA. RISC consists of proteins that scan the binding site on the target mRNA and cleave the miRNA-mRNA duplex. Argonaute2 (Ago2) is the main component of RISC. Out of the five Ago isoforms (Ago1-Ago5), Ago2 is the only one that has endonuclease activity and participates in RNAi in human cells⁷^,⁸^,⁹^,¹⁰. The Ago2-miRNA-RISC complex is the functional unit for miRNA-mediated post-transcriptional mRNA repression¹¹. The Ago2-associated miRNA represents the native state of miRNA in response to intracellular or extracellular signaling. Thus, immunoprecipitation of the endogenous Ago2 provides an excellent opportunity to detect the active, RISC-associated fraction of a specific miRNA as well as the functional assessment of its targets. This assay is superior to the pull-down of endogenous target mRNA with biotinylated miRNA mimics because of unpredictable efficiency of the cellular uptake of biotinylated nucleic acid molecules and their off-target effects.

The Ago2-miRNA-co-IP assay, discussed in this manuscript, was optimized to determine the effects of TGF-β1 on RISC recruitment of miRNAs in immortalized human bronchial epithelial CFBE41o- cells³. Components of the RIP assay kit were used to perform Ago2-miRNA-co-IP assay with modifications in the protocol provided by the manufacturer. A separation method was used to isolate small and large RNA, in which small RNA was used to quantify miRNA with the help of quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) using miRNA-specific stem-looped primers during reverse transcription, followed by PCR using miRNA-specific forward and reverse primers, and TaqMan hydrolysis probes.

Protocol

1. Preparation before experiment Seeding cells Prepare 10% collagen I solution in Minimal Essential Medium (MEM) and add 500 µL to each 24 mm cell culture filter in a 6-well plate. Distribute to cover the entire surface of the filter by rotating gently by hand. Incubate filters under the UV light in the laminar flow hood at room temperature for 30 minutes (min), followed by incubation in cell culture incubator at 37 °C for 1 h. Prepare cell culture medium (MEM…

Representative Results

We have previously shown that TGF-β1 increased the total cellular levels of miR-145-5p, miR-143-5p, and miR-154-5p miRNAs in CFBE41o- cells3. Next, we employed the Ago2-miRNA-co-IP assay to elucidate the functional effects of TGF-β1 on these miRNAs. The RISC recruitment of miR-145-5p, miR-143-5p, and miR-154-5p was studied in CFBE41o- cells stably expressing the wild type (WT)-cystic fibrosis transmembrane conductance regulator (CFTR) or mutant CFTR with the deletion of phenylalanin…

Discussion

The Ago2-miRNA-co-IP assay is designed to investigate the active pool of miRNAs in response to TGF-β1 treatment. The active or RISC-associated miRNAs are important to understand their inhibitory potential for the target mRNA⁴. Panshin et al. recently showed that the immunoprecipitation efficiency of Ago2 and miRNAs may depend on the protocol¹⁶. There are several differences between the protocol here and the above published data. The protocol here was optimized for CFBE…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

We thank John Wakefield from Tranzyme, Inc. (Birmingham, AL) who generated the CFBE41o- cells, and J.P. Clancy from the CFFT who provided the cells. This research was funded by the National Institutes of Health grants R01HL144539 and R56HL127202 (to A.S.-U.), and the Cystic Fibrosis Foundation grant SWIATE18G0.

Materials

100 mM dNTPs (with dTTPs)	Applied Biosystems	4366596
10x Reverse Transcription Buffer (RT Buffer)	Applied Biosystems	4366596
2-propanol	Fisher BioReagents	BP2618-1
2x Laemmli Sample Buffer	Bio-Rad	1610737
7300 Real Time PCR System	Applied Biosystems	4345240
Anti-Ago2 antibody (anti-EIF2C2), mouse monoclonal against human Ago2	Medical & Biological Laboratories Co. Ltd	RN003M
Bovine Albumin Fraction V (7.5% solution)	Thermo Scientific	15260037
Collagen I (Purecol-Type I Bovie collagen solution)	Advanced Biometrix	50005-100mL
DL-Dithiothreitol (DTT)	Sigma	646563-.5ML
DTT	Sigma-Aldrich	646563
Ethanol	Deacon Laboratories	64175
Fetal Bovine Serum	ATLANTA Biologicals	S10350
Goat Anti-Mouse IgG	Bio-Rad	1706516
L-glutamine (200 mM Solution; 29.20 mg/mL)	Corning	25-005-Cl
Mini cell scrapers United Biosystems	Thermo Fisher	MCS-200
Minimal Essential Medium	Thermo Fisher Scientific	11095-080
miRNA specific stem looped RT primers	Applied Biosystems	4427975
Mouse IgG2 control	Dako, Glostrup, Denmark	A0424
MultiScribe Reverse Transcriptase, 50 U/µL	Applied Biosystems	4366596
Nano Drop ND-1000 Spectrophotometer	NanoDrop Technologies, Inc.	E112352
Nuclease-free water	Ambion	AM9937
Opti-MEM (1x) Reduced Serum Medium	Gibco by Life Technologies	11058-021
PBS	Gibco	14190250
Penicillin-streptomycin, Sterile	Sigma-Aldrich	P0781
Pierce Protease Inhibitor Tablets, EDTA-Free	Thermo Scientific	A32955
Protein G agarose beads (Pierce Protein G Plus Agarose)	Thermo Scientific	22851
Puromycin	InvivoGen	ant-pr-1
RiboCluster Profiler RIP-Assay Kit for microRNA	Medical & Biological Laboratories Co. Ltd	RN1005
RNase Inhibitor, 20 U/µL	Applied Biosystems	4366596
TaqMan 2x Universal PCR master mix without AmpErase UNG	Applied Biosystems	4427975
TaqMan miRNA single tube Assay (20x) containing miRNA specific forward/reverse primers and probe	Applied Biosystems	4427975 (assay ID #002278, #002146, and #000477)
TGF-beta1	Sigma	T1654
Transwell filters (24 mm)	Corning Life Sciences Plastic	3412
Veriti 96 Well Thermal Cycler (Model:9902)	Applied Biosystems	4375786

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