Identification of RNA Binding Proteins by UV Cross‐Linking

Ming‐Juan Luo1, Robin Reed1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 27.2
DOI:  10.1002/0471142727.mb2702s63
Online Posting Date:  August, 2003
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Abstract

One of the major focuses of modern biology is to understand the dynamics of RNA‐protein interactions, including factors that interact with mRNA, rRNA, tRNA, snRNA, hnRNA, siRNA, and viral RNA. Identification the direct interactions between proteins and RNA has greatly advanced our knowledge about the function of RNA‐protein machines. UV crosslinking is a straightforward and powerful tool in this endeavor.

     
 
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Table of Contents

  • Basic Protocol 1: UV Cross‐Linking Using a Uniformly Labeled Radioactive RNA
  • Alternate Protocol 1: Immunoprecipitation of UV‐Cross‐Linked Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: UV Cross‐Linking Using a Uniformly Labeled Radioactive RNA

  Materials
  • Linearized (unit 3.1) DNA template for transcription of the RNA of interest
  • 200 mM and 2 M DTT (American Bioanalytical)
  • 0.1% (v/v) Triton X‐100 (Sigma)
  • 1 mM NTPs (unit 3.4)
  • 6.7 mM G(5′)ppp(5′)G cap (NEB)
  • RNasin (Amersham Pharmacia Biotech)
  • High specificity 32P‐labeled nucleotides (NEN):
    • 10 Ci/µl [α‐32P]UTP (3000 Ci/mmol)
    • 10 Ci/µl [α‐32P]ATP (3000 Ci/mmol)
    • 10 Ci/µl [α‐32P]GTP (3000 Ci/mmol)
    • 10 Ci/µl [α‐32P]CTP (3000 Ci/mmol)
  • 50 U/µl T7 or 20 U/µl SP6 RNA polymerase (NEB)
  • 5× transcription buffer (see recipe)
  • 5 mg/ml DNase I (Amersham Pharmacia Biotech)
  • RNA phenol (American Bioanalytical)
  • 5 M ammonium acetate
  • 20 mg/ml glycogen, molecular‐biology grade (Amersham Pharmacia Biotech)
  • 70% and 100% ethanol
  • 12.5 mM ATP
  • 80 mM MgCl 2
  • 0.5 M creatine phosphate (prepare from di‐Tris salt)
  • Splicing dilution buffer (see recipe)
  • HeLa cell nuclear extract (unit 27.3; store at −80°C)
  • Sephacryl S‐500 high resolution resin (Amersham Pharmacia Biotech)
  • 1× gel‐filtration column buffer (see recipe)
  • 10 mg/ml protease‐free RNase A (Amersham Pharmacia Biotech)
  • 100 U/µl RNase T1 (Boehringer Mannheim)
  • 20% (w/v) SDS
  • Acetone
  • 2× protein‐gel sample buffer (see recipe) or 2× SDS sample buffer (unit 10.2)
  • 30°, 37°, 40° (for SP6 transcripts), 65°, and 100°C water baths
  • 1.5‐cm‐diameter × 50‐cm‐long column (89‐ml bed volume)
  • Microcentrifuge tubes with caps removed
  • 254‐nm UV light source (e.g., Sylvania G15T8 lamp)
  • Additional reagents and equipment for packing gel filtration columns (unit 10.9), 1‐D SDS‐PAGE (unit 10.2) or 2‐D SDS PAGE (units 10.3 & 10.4), and autoradiography ( 3.NaN)

Alternate Protocol 1: Immunoprecipitation of UV‐Cross‐Linked Proteins

  • Protein A– or protein G–Sepharose CL‐4B (Amersham Pharmacia Biotech)
  • 1× PBS, pH 7.4 ( appendix 22)
  • Antibody against the protein of interest
  • IP100 buffer (see recipe)
  • SDS
  • Triton X‐100
  • IP150/1 M urea (see recipe)
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Figures

  •   FigureFigure 27.2.1 Isolation of spliceosomes and H complex by gel filtration. A representative gel filtration profile using a Sephacryl S500 column in shown. Peaks: V, void volume, S, spliceosomes, H, H complex; F, free counts.
  •   FigureFigure 27.2.2 General outcome of autoradiograms of typical UV cross‐linking experiments when different parameters (as indicated) are chosen in this protocol. Lane 1, marker; lane 2, body‐labeled RNA, lane 3, site‐specific‐labeled RNA, lane 4, gel‐filtered site‐specific‐labeled RNA; lane 5, gel‐filtered and immunoprecipitated site‐specific‐labeled RNA.

Videos

Literature Cited

   Chiara, M.D., Palandjian, L., Feld Kramer, R., and Reed, R. 1997. Evidence that U5 snRNP recognizes the 3′ splice site for catalytic step II in mammals. E.M.B.O. J. 16:4746‐4759.
   Costas, C., Yuriev, E., Meyer, K.L., Guion, T.S., and Hanna, M.M. 2000. RNA‐protein cross‐linking to AMP residues at internal positions in RNA with a new photocross‐linking ATP analog. Nucl. Acids Res. 28:1849‐1858.
   Hanna, M.M. 1989. Photoaffinity cross‐linking methods for studying RNA‐protein interactions. Methods Enzymol. 180:383‐409.
   Hanna, M.M., Dissinger, S., Williams, B.D., and Colston, J.E. 1989. Synthesis and characterization of 5‐[(4‐azidophenacyl)thio]uridine 5′‐triphosphate, a cleavable photo‐cross‐linking nucleotide analogue. Biochemistry. 28:5814‐20.
   Hanna, M.M., Zhang, Y., Reidling, J.C., Thomas, M.J., and Jou, J. 1993. Synthesis and characterization of a new photocross‐linking CTP analog and its use in photoaffinity labeling E. coli and T7 RNA polymerases. Nucl. Acids Res. 21:2073‐2079.
   Hartley, R., Le Meuth‐Metzinger, V., and Osborne, H.B. 2002. Screening for sequence‐specific RNA‐BPs by comprehensive UV cross‐linking. B.M.C. Mol. Biol. 7:3:8.
   Kwon, Y.K. and Hecht, N.B. 1991. Cytoplasmic protein binding to highly conserved sequences in the 3′ untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells. Proc. Natl. Acad. Sci. U.S.A. 88:3584‐3588.
   Leibold, E.A. and Munro, H.N. 1988. Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5′ untranslated region of ferritin heavy‐ and light‐subunit mRNAs. Proc. Natl. Acad. Sci. U.S.A. 85:2171‐2175.
   Lynch, K.W. and Maniatis, T. 1996. Assembly of specific SR protein complexes on distinct regulatory elements of the Drosophila doublesex splicing enhancer. Genes Dev. 10:2089‐2101.
   Marczinovits, I. and Molnar, J. 1982. Ultraviolet light‐induced cross‐linking of HnRNA to informofer proteins in vitro. Mol. Biol. Rep. 8:111‐116.
   Moore, M.J. and Sharp, P.A. 1992. Site‐specific modification of pre‐mRNA: The 2′‐hydroxyl groups at the splice sites. Science 256:992‐997.
   Ping, Y.H., Liu, Y., Wang, X., Neenhold, H.R., and Rana, T.M. 1997. Dynamics of RNA‐protein interactions in the HIV‐1 Rev‐RRE complex visualized by 6‐thioguanosine‐mediated photocross‐linking. RNA. 3:850‐860.
   Reed, R. and Chiara, M. D. 1999. Identification of RNA‐protein contacts within functional ribonucleoprotein complexes by RNA site‐specific labeling and UV cross‐linking. Methods 18:3‐12.
   Tanner, N.K., Hanna, M.M., and Abelson, J. 1988. Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues. Biochemistry 27:8852‐8861.
   Teigelkamp, S., Newman, A.J., and Beggs, J.D. 1995. Extensive interactions of PRP8 protein with the 5′ and 3′ splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA. E.M.B.O. J. 14:2602‐2612.
   Umen, J.G. and Guthrie, C. 1995. Prp16p, Slu7p, and Prp8p interact with the 3′ splice site in two distinct stages during the second catalytic step of pre‐mRNA splicing. RNA. 1:584‐597.
   Woody, A.Y., Vader, C.R., Woody, R.W., and Haley, B.E. 1984. Photoaffinity labeling of DNA‐dependent RNA polymerase from Escherichia coli with 8‐azidoadenosine 5′‐triphosphate. Biochemistry. 23:2843‐2848.
   Woody, A.Y., Evans, R.K., and Woody, R.W. 1988. Characterization of a photoaffinity analog of UTP, 5‐azido‐UTP for analysis of the substrate binding site on E. coli RNA polymerase. Biochem. Biophys. Res. Commun. 150:917‐924.
   Wyatt, J.R., Sontheimer, E.J., and Steitz, J.A. 1992. Site‐specific cross‐linking of mammalian U5 snRNP to the 5′ splice site before the first step of pre‐mRNA splicing. Genes Dev. 6:2542‐2553.
   Xiang, R.H. and Lee, J.C. 1989. Identification of proteins cross‐linked to RNA in 40S ribosomal subunits of Saccharomyces cerevisiae. Biochimie. 71:1201‐1204.
Key References
   Hanna, M.M. 1989. See above.
  A seminal paper describing the use of several photoreactive nucleotide analogues in the UV cross‐linking assay.
   Hartley et al., 2002. See above.
  Describes the ups and downs of the different choices of radiolabeled nucleotides in the UV cross‐linking assay.
   Reed and Chiara, 1999. See above.
  Contains a very detailed description of the UV cross‐linking assay combined with site specific labeled RNA to study RNA‐protein interactions in functional spliceosomes.
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