Purification of Functional RNA‐Protein Complexes using MS2‐MBP

Zhaolan Zhou1, Robin Reed1

1 Children's Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 27.3
DOI:  10.1002/0471142727.mb2703s63
Online Posting Date:  August, 2003
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Biological machines composed of RNAs and proteins play essential roles in many biological processes. To better understand the mechanism and function of these machines, it is critical to isolate them in a highly purified and functional form. A method for isolating functional RNA‐protein complexes assembled in vitro is described. The approach combines gel filtration and an affinity‐chromatography strategy using the bacteriophage MS2 coat protein, which binds to a specific RNA‐hairpin structure. Using this method, highly purified and functional human spliceosomes have been isolated. The purified spliceosome preparation is used to determine the protein components of the spliceosome by mass spectrometry and to examine the structure of the spliceosome by electron microscopy.

Keywords: MS2‐MBP; RNA‐protein complex; ribonucleoprotein complex; spliceosome; affinity purification

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1:

  Materials
  • E.coli strain (e.g., DH5α) containing plasmid expressing MS2‐MBP fusion protein (unit 16.6)
  • Plasmid DNA encoding desired RNA and MS2 binding sites
  • Buffer A: 10 mM sodium phosphate buffer, pH 7.0 ( appendix 22), filtered and degassed
  • Buffer B: 10 mM sodium phosphate buffer, pH 7.0 ( appendix 22)/1 M NaCl, filtered and degassed
  • Dialysis buffer: 10% (v/v) glycerol in PBS (see appendix 22 for PBS)
  • [32P]UTP (sp. act., 800 Ci/mmol; unit 3.4)
  • Splicing dilution buffer (see recipe)
  • 1.5% agarose gel (unit 2.5)
  • 12.5 mM ATP
  • 80 mM MgCl 2
  • 0.5 M creatine phosphate
  • 10 mg/ml HeLa cell nuclear extract (unit 12.1)
  • Gel filtration buffer (see recipe)
  • Amylose resin (New England Biolabs)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Maltose elution buffer (see recipe)
  • HiTrap Heparin HP column (Amersham Biosciences) and FPLC system (unit 10.10) with gradient maker
  • Dialysis cassettes (MWCO 10,000; Pierce)
  • Centricon‐50 centrifugal concentrator (Amicon)
  • 50‐ml polypropylene tubes (e.g., Falcon)
  • Packed 1.5/50 cm Sephacryl S‐500 HR column and a gel‐filtration system (unit 10.9)
  • Scintillation counter capable of Cerenkov counting
  • Data graphing software: e.g., Microsoft Excel or Cricket Graph from Computer Associates International
  • End‐over‐end rotator
  • Additional reagents and equipment for purification of MS2‐MBP protein from E. coli (unit 16.6), dialysis ( appendix 3C), preparation of radiolabeled RNA (unit 14.3, Support Protocol 1), mobility‐shift RNA‐binding assay (unit 12.2), agarose gel electrophoresis (unit 2.5), and gel‐filtration chromatography (unit 10.9)
NOTE: All of the reagents should be RNase free (unit 4.1) and all procedures involving RNA‐protein complex isolation should be carried out at 4°C or on ice.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bardwell, V.J. and Wickens, M. 1990. Purification of RNA and RNA‐protein complexes by an R17 coat protein affinity method. Nucl. Acids Res. 18:6587‐6594.
   Bennett, M., Michaud, S., Kingston, J. and Reed, R. 1992. Protein components specifically associated with prespliceosomes and spliceosome complexes. Genes Dev. 6:1986‐2000.
   Carey, J., Cameron, V., de Haseth, P.L., and Uhlenbeck, O.C. 1983. Sequence‐specific interaction of R17 coat protein with its ribonucleic acid binding site. Biochemistry 22:2601‐2610.
   Das, R., Zhou, Z. and Reed, R. 2000. Functional association of U2 snRNP with the ATP‐independent spliceosomal complex E. Mol. Cell 5: 779‐787.
   Graveley, B.R. and Maniatis, T. 1998. Arginine/Serine‐rich domains of SR proteins can function as activators of pre‐mRNA splicing. Mol. Cell 1:765‐771.
   LeCuyer, K.A., Behlen, L.S. and Uhlenbeck, O.C. 1995. Mutants of the bacteriophage MS2 coat protein that alter its cooperative binding to RNA. Biochemistry 34:10600‐10606.
   Reed, R. 1990. Protein composition of mammalian spliceosomes assembled in vitro. Proc. Natl. Acad. Sci. U.S.A. 87: 8031‐8035.
   Zhou, Z., Luo, M‐J., Straesser, K., Katahira, J., Hurt, E., and Reed, R. 2000. The protein Aly links pre‐messenger RNA splicing to nuclear export in metazoans. Nature 407:401‐405.
   Zhou, Z., Sim, J., Griffith, J., and Reed, R. 2002a. Purification and electron microscopic visualization of functional human spliceosomes. Proc. Natl. Acad. Sci. U.S.A. 99:12203‐12207.
   Zhou, Z., Licklider, L.J., Gygi, S.P., and Reed, R. 2002b. Comprehensive proteomic analysis of the human spliceosome. Nature 419:182‐185.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library