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Molecular and Cellular Biology, July 2006, p. 5528-5543, Vol. 26, No. 14
0270-7306/06/$08.00+0     doi:10.1128/MCB.00582-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Protein Composition and Electron Microscopy Structure of Affinity-Purified Human Spliceosomal B Complexes Isolated under Physiological Conditions

Jochen Deckert,1 Klaus Hartmuth,1 Daniel Boehringer,2,{dagger} Nastaran Behzadnia,1 Cindy L. Will,1 Berthold Kastner,1 Holger Stark,2 Henning Urlaub,3 and Reinhard Lührmann1*

Department of Cellular Biochemistry,1 3D Electron Cryomicroscopy Group,2 Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany3

Received 3 April 2006/ Accepted 19 April 2006

The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including ~50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 Å and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.

* Corresponding author. Mailing address: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany. Phone: 49 551 2011407. Fax: 49 551 2011197. E-mail: Reinhard.Luehrmann{at}mpi-bpc.mpg.de.

{dagger} Present address: Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology, CH-8093 Zürich, Switzerland.

Molecular and Cellular Biology, July 2006, p. 5528-5543, Vol. 26, No. 14
0270-7306/06/$08.00+0     doi:10.1128/MCB.00582-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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