RNA Immunoprecipitation for Determining RNA‐Protein Associations In Vivo

Chris Gilbert1, Jesper Q. Svejstrup1

1 Cancer Research UK, London Research Institute, Clare Hall Laboratories, Hertfordshire, U.K.
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 27.4
DOI:  10.1002/0471142727.mb2704s75
Online Posting Date:  August, 2006
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Similar to chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP) can be used to detect the association of individual proteins with specific nucleic acid regions, in this case on RNA. Live cells are treated with formaldehyde to generate protein‐RNA cross‐links between molecules that are in close proximity in vivo. RNA sequences that cross‐link with a given protein are isolated by immunoprecipitation of the protein, and reversal of the formaldehyde cross‐linking permits recovery and quantitative analysis of the immunoprecipitated RNA by reverse transcription PCR. The basics of RIP are very similar to those of ChIP, but with some important caveats. This unit describes the RIP procedure for Saccharomyces cerevisiae. Although the corresponding steps for metazoan cells have not yet been worked out, it is likely that the yeast procedure can easily be adapted for use in other organisms.

Keywords: RNA immunoprecipitation; RIP; chromatin immunoprecipitation; ChIP; Transcription; RNA metabolism

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Table of Contents

  • Basic Protocol 1: RNA Immunoprecipitation in Yeast Cells
  • Alternate Protocol 1: Analysis by Real‐Time PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: RNA Immunoprecipitation in Yeast Cells

  • Saccharomyces cerevisiae cells to be studied (see Chapter 13)
  • 37% formaldehyde (store up to 1 year at room temperature)
  • 2 M glycine, sterilized by autoclaving
  • Tris‐buffered saline (TBS; appendix 22), ice cold
  • FA lysis buffer (see recipe), ice cold and room temperature
  • 40 U/µl RNasin (Promega)
  • ∼0.5‐mm‐diameter silica‐zirconia (preferably BioSpec) or glass beads
  • Ice/salt mixture in beaker for cooling
  • MgCl 2
  • CaCl 2
  • 20 mg/ml RNase‐free DNase I (Sigma)
  • 0.5 M EDTA ( appendix 22)
  • Primary antibody against protein or epitope of interest
  • 50% (v/v) protein A–Sepharose beads (Amersham Biosciences or equivalent) in FA lysis buffer containing 1 mg/ml BSA
  • FA lysis buffer (see recipe) containing 1 mg/ml BSA
  • FA500 (see recipe)
  • LiCl wash solution (see recipe)
  • TE/100 mM NaCl (see recipe)
  • ChIP elution buffer (see recipe)
  • 5 M NaCl, sterilized by autoclaving
  • 20 mg/ml proteinase K (Roche) in TBS/50% glycerol (store up to 1 year at −20°C)
  • Acid‐equilibrated 5:1 phenol/chloroform, pH 4.7 (Sigma, cat. no. P1944)
  • Phase Lock Gel, Heavy (Eppendorf)
  • 3 M sodium acetate, pH 5.5 ( appendix 22)
  • Glycogen
  • Absolute ethanol, ice cold
  • 70% ethanol
  • TE buffer, pH 7.5 ( appendix 22)
  • Titan One‐Tube RT‐PCR kit (Roche)
  • 6% acrylamide/bisacrylamide (19:1) nondenaturing PAGE gel prepared in TBE buffer (see unit 2.7)
  • SYBR Green (Molecular Probes)
  • 500‐ml Erlenmeyer flask
  • Platform rocker
  • 50‐ml conical centrifuge tubes (e.g., Falcon)
  • Refrigerated centrifuge
  • 1.5‐ml (nonstick) microcentrifuge tubes, certified RNase‐free
  • FastPrep benchtop cell disruptor (Qbiogene)
  • Hypodermic needle
  • 2‐ml microcentrifuge tubes
  • 15‐ml conical polypropylene centrifuge tubes, disposable (e.g., Falcon)
  • Sonicator with microtip probe (e.g., Branson Sonifier 250)
  • End‐over‐end rotator
  • Spin‐X microcentrifuge tube filters (Corning, available, e.g., from Sigma)
  • 42° (optional) and 65°C water baths
  • Thermal cycler
  • Additional reagents and equipment for growth of Saccharomyces cerevisiae cultures (units 13.1& 13.2), determining chromatin fragment size (unit 21.3), phenol/chloroform extraction and ethanol precipitation (unit 2.1), primer design for ChIP experiments (unit 21.3), oligonucleotide synthesis (unit 2.11), PCR (units 15.1& 15.7), nondenaturing polyacrylamide gel electrophoresis (unit 2.7), and agarose gel electrophoresis and ethidium bromide staining of gels (unit 2.5)
NOTE: Remember that this procedure is concerned with detecting RNA; therefore, great care has to be taken to avoid its degradation during handling. After harvesting of cells, use RNase‐free tubes as indicated, pipet tips with aerosol‐barrier filters, and solutions prepared with nuclease‐free water (Ambion). Keep samples on ice.

Alternate Protocol 1: Analysis by Real‐Time PCR

  • 2× SYBR Green QPCR mix (Abgene AB‐1162; contains DNA polymerase)
  • Multiscribe reverse transcriptase (Applied Biosystems)
  • ABI Prism 7000 Sequence Detection System, or equivalent
  • SYBR Green QPCR mix (Abgene AB‐1162)
  • Software for analyzing PCR primers and products
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Literature Cited

   Gilbert, C., Kristjuhan, A., Winkler, G.S., and Svejstrup, J.Q. 2004. Elongator interactions with nascent mRNA revealed by RNA immunoprecipitation. Mol. Cell 14:457‐464.
   Gorlich, D., Kraft, R., Kostka, S., Vogel, F., Hartmann, E., Laskey, R.A., Mattaj, I.W., and Izaurralde, E. 1996. Importin provides a link between nuclear protein import and U snRNA export. Cell 87:21‐32.
   Huang, B., Johansson, M.J., and Bystrom, A.S. 2005. An early step in wobble uridine tRNA modification requires the Elongator complex. RNA 11:424‐436.
   Huertas, P. and Aguilera, A. 2003. Co‐transcriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription‐associated recombination. Mol. Cell 12:711‐721.
   Hurt, E., Luo, M.J., Rother, S., Reed, R., and Strasser, K. 2004. Cotranscriptional recruitment of the serine‐arginine‐rich (SR)‐like proteins Gbp2 and Hrb1 to nascent mRNA via the TREX complex. Proc. Natl. Acad. Sci. U.S.A. 101:1858‐1862.
   Izaurralde, E., Lewis, J., Gamberi, C., Jarmolowski, A., McGuigan, C., and Mattaj, I.W. 1995. A cap‐binding protein complex mediating U snRNA export. Nature 376:709‐712.
   Kristjuhan, A. and Svejstrup, J.Q. 2004. Evidence for distinct mechanisms facilitating transcript elongation through chromatin in vivo. EMBO J. 23:4243‐4252.
   Motamedi, M.R., Verdel, A., Colmenares, S.U., Gerber, S.A., Gygi, S.P., and Moazed, D. 2004. Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119:789‐802.
   Robert, F., Pokholok, D.K., Hannett, N.M., Rinaldi, N.J., Chandy, M., Rolfe, A., Workman, J.L., Gifford, D.K., and Young, R.A. 2004. Global position and recruitment of HATs and HDACs in the yeast genome. Mol. Cell 16:199‐209.
   Svejstrup, J.Q. 2003. Keeping RNA and DNA apart during transcription. Mol. Cell 12:538‐539.
Key References
   Gilbert et al., 2004. See above.
  Describes the technique from which the was adapted.
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