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In This Article

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

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 cond.......

Representative 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.......

Acknowledgements

This work was supported by the NIH grant GM120209 (GS). The authors thank the OSUCCC Genomics Shared Resources Core for their services (CCC Support Grant NCI P30 CA16058).

....

Materials

NameCompanyCatalog NumberComments
Anti-FLAG Affinity GelSigmaA2220
ATP, [γ-32P]- 3000Ci/mmol 10mCi/ml EasyTide, 250µCiPerkinElmerBLU502A250UC
BD Disposable Syringes with Luer-Lok Tips (200)Fisher14-823-435
Betaine 5MSigmaB0300
biotin-dATPTriLinkN-5002
biotin-dCTPPerkin ElmerNEL540001EA
Branson Sonifier, Model SSE-1Branson
CircLigase IVWR76081-606ssDNA ligase I
DMEM, High GlucoseThermoFisher11995-065
DNA load buffer NEBNEB
Dynabeads Protein ALifeTech10002D
Flp-In-T-REx 293 Cell LineThermoFisherR78007
GeneRuler Low Range DNA LadderThermoScientificFERSM1203
Hygromycin BThermoFisher10687010
Mini-PROTEAN TBE Gel 10 wellBio-Rad4565013
Mini-PROTEAN TBE-Urea GelBio-Rad4566033
miRCAT-33 adapter 5′-TGGAATTCTCGGGTGCCAAGGddC-3′Anythis protocol is only compatible with the Illumina sequencing platform
Mirus transIT-X2 transfection reagentMirusMIR 6004
Mth RNA ligaseNEBE2610S
PE1.0 5′-AATGATACGGCGACCACCGAGATCTACACT
CTTTCCCTACACGACGCTCTTCCGATC*T-3′
Anythis protocol is only compatible with the Illumina sequencing platform
PE2.0 5′-CAAGCAGAAGACGGCATACGAGATCGGTCTC
GGCATTCCTGCTGAACCGCTCTTCCGATC*T-3′
Anythis protocol is only compatible with the Illumina sequencing platform
Phenol/Chloroform/Isoamyl Alcohol (25:24:1, pH 6.7, 100ml)FisherBP1752I-100
Purple Gel Loading Dye (6x)NEBNEB #7025
Q5 DNA PolymeraseNEBM0491S/L
RNase I, E. coli, 1000 unitsEppicenterN6901K
SPIN-X columnCorningCLS8160-24EA
Streptavidin beadsThermoFisher60210
Superscript III (SSIII)ThermoScientific18080044reverse transcriptase enzyme
SybrGoldThermoFisherS11494gold nucleic acid gel stain
T4 Polynucleotide Kinase-2500UNEBM0201L
T4RNL2 Tr. K227QNEBM0351S
TetracyclineSigma87128
Thermostable 5´ App DNA/RNA LigaseNEBM0319S
TruSeq_SE1 5′-pGGCACTANNNNNAGATCGGAAGA
GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC
TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′
Anythis protocol is only compatible with the Illumina sequencing platform
TruSeq_SE10 5′-pGGTGTTCNNNNNAGATCGGAAG
AGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCT
CTTCCGATCTCCTTGGCACCCGAGAATTCCA-3′
Anythis protocol is only compatible with the Illumina sequencing platform
TruSeq_SE11 5′-pGGTAAGTNNNNNAGATCGGAA
GAGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC
TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′
Anythis protocol is only compatible with the Illumina sequencing platform
TruSeq_SE12 5′-pGGAGATGNNNNNAGATCGGAAGA
GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC
TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′
Anythis protocol is only compatible with the Illumina sequencing platform
TruSeq_SE2 5′-pGGGTAGCNNNNNAGATCGGAAGAG
CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCT
CTTCCGATCTCCTTGGCACCCGAGAATTCCA-3′
Anythis protocol is only compatible with the Illumina sequencing platform
TruSeq_SE35′-pGGTCGATNNNNNAGATCGGAAG
AGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCT
CTTCCGATCTCCTTGGCACCCGAGAATTCCA-3′
Anythis protocol is only compatible with the Illumina sequencing platform
TruSeq_SE4 5′-pGGCCTCGNNNNNAGATCGGAAGA
GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC
TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′
Anythis protocol is only compatible with the Illumina sequencing platform
TruSeq_SE5 5′-pGGTGACANNNNNAGATCGGAAGA
GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC
TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′
Anythis protocol is only compatible with the Illumina sequencing platform
TruSeq_SE6 5′-pGGTAGACNNNNNAGATCGGAAGAG
CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCTTC
CGATCTCCTTGGCACCCGAGAATTCCA-3′
Anythis protocol is only compatible with the Illumina sequencing platform
TruSeq_SE7 5′-pGGGCCCTNNNNNAGATCGGAAG
AGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCT
TCCGATCTCCTTGGCACCCGAGAATTCCA-3′
Anythis protocol is only compatible with the Illumina sequencing platform
TruSeq_SE8 5′-pGGATCGGNNNNNAGATCGGAAGAG
CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCTT
CCGATCTCCTTGGCACCCGAGAATTCCA-3′
Anythis protocol is only compatible with the Illumina sequencing platform
TruSeq_SE9 5′-pGGACTGANNNNNAGATCGGAAGAG
CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCTTC
CGATCTCCTTGGCACCCGAGAATTCCA-3′
Anythis protocol is only compatible with the Illumina sequencing platform
Typhoon 5 Bimolecular ImagerGE Healthcare Life Science29187191

References

  1. Karousis, E. D., Nasif, S., Mühlemann, O. Nonsense-mediated mRNA decay: novel mechanistic insights and biological impact. Wiley Interdisciplinary Reviews: RNA. 7 (5), 661-682 (2016).
  2. Ivanov, P. V., Gehring, N. H., Kunz, J. B., Hentze, M. W., Kulozik, A. E.

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