Previous Article | Next Article 
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,
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.
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.
This article has been cited by other articles:
-
Little, J. T., Jurica, M. S.
(2008). Splicing Factor SPF30 Bridges an Interaction between the Prespliceosome Protein U2AF35 and Tri-small Nuclear Ribonucleoprotein Protein hPrp3. J. Biol. Chem.
283: 8145-8152
[Abstract]
[Full Text]
-
Pearson, J. L., Robinson, T. J., Munoz, M. J., Kornblihtt, A. R., Garcia-Blanco, M. A.
(2008). Identification of the Cellular Targets of the Transcription Factor TCERG1 Reveals a Prevalent Role in mRNA Processing. J. Biol. Chem.
283: 7949-7961
[Abstract]
[Full Text]
-
Penn, J. K. M., Graham, P., Deshpande, G., Calhoun, G., Chaouki, A. S., Salz, H. K., Schedl, P.
(2008). Functioning of the Drosophila Wilms'-Tumor-1-Associated Protein Homolog, Fl(2)d, in Sex-Lethal-Dependent Alternative Splicing. Genetics
178: 737-748
[Abstract]
[Full Text]
-
Novoyatleva, T., Heinrich, B., Tang, Y., Benderska, N., Butchbach, M. E.R., Lorson, C. L., Lorson, M. A., Ben-Dov, C., Fehlbaum, P., Bracco, L., Burghes, A. H.M., Bollen, M., Stamm, S.
(2008). Protein phosphatase 1 binds to the RNA recognition motif of several splicing factors and regulates alternative pre-mRNA processing. Hum Mol Genet
17: 52-70
[Abstract]
[Full Text]
-
Alberstein, M., Amit, M., Vaknin, K., O'Donnell, A., Farhy, C., Lerenthal, Y., Shomron, N., Shaham, O., Sharrocks, A. D., Ashery-Padan, R., Ast, G.
(2007). Regulation of transcription of the RNA splicing factor hSlu7 by Elk-1 and Sp1 affects alternative splicing. RNA
13: 1988-1999
[Abstract]
[Full Text]
-
Heinze, M., Kofler, M., Freund, C.
(2007). Investigating the functional role of CD2BP2 in T cells. Int Immunol
19: 1313-1318
[Abstract]
[Full Text]
-
Kuhn-Holsken, E., Dybkov, O., Sander, B., Luhrmann, R., Urlaub, H.
(2007). Improved identification of enriched peptide RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry. Nucleic Acids Res
35: e95-e95
[Abstract]
[Full Text]
-
Gullerova, M., Barta, A., Lorkovic, Z. J.
(2007). Rct1, a Nuclear RNA Recognition Motif-Containing Cyclophilin, Regulates Phosphorylation of the RNA Polymerase II C-Terminal Domain. Mol. Cell. Biol.
27: 3601-3611
[Abstract]
[Full Text]
-
Merz, C., Urlaub, H., Will, C. L., Luhrmann, R.
(2007). Protein composition of human mRNPs spliced in vitro and differential requirements for mRNP protein recruitment. RNA
13: 116-128
[Abstract]
[Full Text]
Copyright © 2006 by the American Society for Microbiology. All rights reserved.