Agarose Gel Separation/Isolation of RNA‐Protein Complexes

Chung‐Sheng Brian Lee1, Rita Das1, Robin Reed1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 27.1
DOI:  10.1002/0471142727.mb2701s63
Online Posting Date:  August, 2003
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Abstract

In many methods currently used to analyze RNA‐protein complexes, high salt or other stringent treatments are required for reducing nonspecific interactions and resolving the complex of interest. RNA‐protein complexes often dissociate on native polyacrylamide gels and can only be detected on density gradients or by gel filtration. Agarose gel electrophoresis provides an alternative method that is simple, rapid, and can have high resolution of RNA‐protein complexes. Moreover, the use of low‐melting point agarose for the fractionation readily allows for the isolation of the RNA species in each complex detected on the native gel.

Keywords: RNA‐protein complex; low‐melting‐point agarose; agarose minigel system

     
 
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Table of Contents

  • Basic Protocol 1: Electrophoretic Isolation of RNA‐Protein Complexes
  • Support Protocol 1: Elution of RNA from Gel‐Fixed RNA‐Protein Complexes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Electrophoretic Isolation of RNA‐Protein Complexes

  Materials
  • DNA encoding desired sequence for RNA transcription (units 1.5 1.7)
  • Reaction mixture (e.g., HeLa cell nuclear extracts, unit 27.3)
  • 10× sample loading dye or 10× heparin loading dye (see reciperecipes)
  • Low‐melting‐point agarose (Life Technologies)
  • 0.5× TBE electrophoresis buffer ( appendix 22)
  • Gel‐fixing solution: 10% acetic acid/10% methanol
  • Additional reagents and equipment for preparing 32P‐labeled RNA transcripts (unit 27.3), electrophoresis using agarose minigels (unit 2.5), and autoradiography ( appendix 3A)
NOTE: Extreme caution should be taken to avoid RNase contamination. The experimenter should always wear gloves, and all the tubes and tips that come into contact with the sample should be certified RNase free; however, DEPC treatment (unit 4.1) of solutions and apparatus is not necessary.

Support Protocol 1: Elution of RNA from Gel‐Fixed RNA‐Protein Complexes

  Materials
  • Gel containing bands of interest (see protocol 1)
  • 2× PK buffer (see recipe)
  • Proteinase K
  • RNase‐free phenol, pH 5.2
  • 20 mg/ml glycogen, molecular‐biology grade
  • 100% ethanol
  • Formamide loading dye (see recipe)
  • Additional reagents and equipment for autoradiography ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Chau, K. and Reed, R. 1999. The RNA splicing factor hSlu7 is required for correct 3′ splice‐site choice. Nature 402:207‐210.
   Das, R. and Reed, R. 1999. Resolution of the mammalian E complex and the ATP‐dependent spliceosomal complexes on native agarose mini‐gels. RNA 5:1504‐1508.
   Das, R., Zhou, Z., and Reed, R. 2000. Functional association of U2 snRNP with the ATP‐independent spliceosomal complex E. Molecular Cell 5:779‐787.
   Grabowski, P.J., Seiter, S.R., and Sharp, P.A. 1985. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell 42:345‐353.
   Konarska, M.M. and Sharp, P.A. 1986. Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs. Cell 46:845‐855.
   Konarska, M.M., Grabowski, P.J., Padgett, R.A., and Sharp, P.A. 1985. Characterization of the branch site in lariat RNAs produced by splicing of mRNA precursors. Nature 313:552‐557.
   Luo, M.J. and Reed, R. 1999. Splicing is required for rapid and efficient mRNA export in metazoans. Proc. Natl. Acad. Sci. U.S.A. 96:14937‐14942.
   Michaud, S. and Reed, R. 1991. An ATP‐independent complex commits pre‐mRNA to the mammalian spliceosome assembly pathway. Genes & Dev. 5:2534‐2546.
   Pikielny, C.W., Rymond, B.C., and Rosbash, M. 1986. Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes. Nature 324:341‐345.
   Reed, R. 1990. Protein composition of mammalian spliceosomes assembled in vitro. Proc. Natl. Acad. Sci. U.S.A. 87:8031‐8035.
   Seraphin, B. and Rosbash, M. 1989. Identification of functional U1 snRNA‐pre‐mRNA complexes committed to spliceosome assembly and splicing. Cell 59:349‐358.
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