Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Summary

Here, we present a protocol to enrich endogenous RNA binding sites or "footprints" of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

Abstract

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT's advantages and applications and discuss some of its limitations.

Introduction

Within cells, RNA exists in complex with proteins to form RNA:protein complexes (RNPs). RNPs are assembled around RNA binding proteins (RBPs, those that directly bind RNA) but also comprise of non-RBPs (those that bind RBPs but not RNA), and are often dynamic in nature. RBPs and their cofactors function collectively within RNPs to execute regulatory functions. For example, in the nonsense-mediated mRNA decay (NMD) pathway, the UPF proteins (UPF1, UPF2, and UPF3b) recognize the prematurely terminated ribosome. Each of the UPF proteins can bind to RNA, but it is only when they assemble together that an active NMD complex begins to form. Within this complex, UPF1 is furt....

Protocol

1. Establishment of Stable HEK293 Cell Lines Expressing Tetracycline-inducible FLAG-tagged Protein of Interest (POI)

1. Seed HEK293 cells with a stably integrated Flp recombination target (FRT) site at a density of 10 x 10⁴ cells/mL in growth medium (Dulbecco's modified Eagle's medium [DMEM] + 10% fetal bovine serum [FBS] + 1% penicillin-streptomycin [penn/strep]) in 6-well plates. Allow cells to grow overnight in a humidified incubator at 37 °C and 5% CO₂ (standard growth conditions for all subsequent steps).
2. The next day, cells should be ~70% confluent. According to transfection reagent protocol, transfect the FR....

Results

A successful RIPiT will result in the immunoprecipitation of both proteins of interest and other known interacting proteins, and the absence of non-interacting proteins. As seen in Figure 3A, both Magoh and EIF4AIII were detected in the RIPiT elution, but HNRNPA1 was not (lane 6). In parallel, RNA footprints that have co-purified with the RNP complexes was detected via autoradiography (Figure 3B) or bioanalyzer (

Discussion

We discuss here some key considerations to successfully perform RIPiT. Foremost, individual IPs must be optimized to achieve highest possible efficiency at each step. The amount of FLAG agarose beads for the input number of cells described here has proven to be robust for a wide range of proteins we have tested. As only a small fraction of partner proteins is co-immunoprecipitated with the FLAG protein, the amount of antibody needed for efficient second IP is usually low (less than 10 µg). Small-scale RIPiT (from on.......

Materials

Name	Company	Catalog Number	Comments
Anti-FLAG Affinity Gel	Sigma	A2220
ATP, [γ-32P]- 3000Ci/mmol 10mCi/ml EasyTide, 250µCi	PerkinElmer	BLU502A250UC
BD Disposable Syringes with Luer-Lok Tips (200)	Fisher	14-823-435
Betaine 5M	Sigma	B0300
biotin-dATP	TriLink	N-5002
biotin-dCTP	Perkin Elmer	NEL540001EA
Branson Sonifier, Model SSE-1	Branson
CircLigase I	VWR	76081-606	ssDNA ligase I
DMEM, High Glucose	ThermoFisher	11995-065
DNA load buffer NEB	NEB
Dynabeads Protein A	LifeTech	10002D
Flp-In-T-REx 293 Cell Line	ThermoFisher	R78007
GeneRuler Low Range DNA Ladder	ThermoScientific	FERSM1203
Hygromycin B	ThermoFisher	10687010
Mini-PROTEAN TBE Gel 10 well	Bio-Rad	4565013
Mini-PROTEAN TBE-Urea Gel	Bio-Rad	4566033
miRCAT-33 adapter 5′-TGGAATTCTCGGGTGCCAAGGddC-3′	Any		this protocol is only compatible with the Illumina sequencing platform
Mirus transIT-X2 transfection reagent	Mirus	MIR 6004
Mth RNA ligase	NEB	E2610S
PE1.0 5′-AATGATACGGCGACCACCGAGATCTACACT CTTTCCCTACACGACGCTCTTCCGATC*T-3′	Any		this protocol is only compatible with the Illumina sequencing platform
PE2.0 5′-CAAGCAGAAGACGGCATACGAGATCGGTCTC GGCATTCCTGCTGAACCGCTCTTCCGATC*T-3′	Any		this protocol is only compatible with the Illumina sequencing platform
Phenol/Chloroform/Isoamyl Alcohol (25:24:1, pH 6.7, 100ml)	Fisher	BP1752I-100
Purple Gel Loading Dye (6x)	NEB	NEB #7025
Q5 DNA Polymerase	NEB	M0491S/L
RNase I, E. coli, 1000 units	Eppicenter	N6901K
SPIN-X column	Corning	CLS8160-24EA
Streptavidin beads	ThermoFisher	60210
Superscript III (SSIII)	ThermoScientific	18080044	reverse transcriptase enzyme
SybrGold	ThermoFisher	S11494	gold nucleic acid gel stain
T4 Polynucleotide Kinase-2500U	NEB	M0201L
T4RNL2 Tr. K227Q	NEB	M0351S
Tetracycline	Sigma	87128
Thermostable 5´ App DNA/RNA Ligase	NEB	M0319S
TruSeq_SE1 5′-pGGCACTANNNNNAGATCGGAAGA GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′	Any		this protocol is only compatible with the Illumina sequencing platform
TruSeq_SE10 5′-pGGTGTTCNNNNNAGATCGGAAG AGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCT CTTCCGATCTCCTTGGCACCCGAGAATTCCA-3′	Any		this protocol is only compatible with the Illumina sequencing platform
TruSeq_SE11 5′-pGGTAAGTNNNNNAGATCGGAA GAGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′	Any		this protocol is only compatible with the Illumina sequencing platform
TruSeq_SE12 5′-pGGAGATGNNNNNAGATCGGAAGA GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′	Any		this protocol is only compatible with the Illumina sequencing platform
TruSeq_SE2 5′-pGGGTAGCNNNNNAGATCGGAAGAG CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCT CTTCCGATCTCCTTGGCACCCGAGAATTCCA-3′	Any		this protocol is only compatible with the Illumina sequencing platform
TruSeq_SE35′-pGGTCGATNNNNNAGATCGGAAG AGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCT CTTCCGATCTCCTTGGCACCCGAGAATTCCA-3′	Any		this protocol is only compatible with the Illumina sequencing platform
TruSeq_SE4 5′-pGGCCTCGNNNNNAGATCGGAAGA GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′	Any		this protocol is only compatible with the Illumina sequencing platform
TruSeq_SE5 5′-pGGTGACANNNNNAGATCGGAAGA GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′	Any		this protocol is only compatible with the Illumina sequencing platform
TruSeq_SE6 5′-pGGTAGACNNNNNAGATCGGAAGAG CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCTTC CGATCTCCTTGGCACCCGAGAATTCCA-3′	Any		this protocol is only compatible with the Illumina sequencing platform
TruSeq_SE7 5′-pGGGCCCTNNNNNAGATCGGAAG AGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCT TCCGATCTCCTTGGCACCCGAGAATTCCA-3′	Any		this protocol is only compatible with the Illumina sequencing platform
TruSeq_SE8 5′-pGGATCGGNNNNNAGATCGGAAGAG CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCTT CCGATCTCCTTGGCACCCGAGAATTCCA-3′	Any		this protocol is only compatible with the Illumina sequencing platform
TruSeq_SE9 5′-pGGACTGANNNNNAGATCGGAAGAG CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCTTC CGATCTCCTTGGCACCCGAGAATTCCA-3′	Any		this protocol is only compatible with the Illumina sequencing platform
Typhoon 5 Bimolecular Imager	GE Healthcare Life Science	29187191