Article
- The EMBO Journal (2007) 26, 1737 - 1748
- doi:10.1038/sj.emboj.7601631
Published online: 1 March 2007
Subject Categories:
Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes
Nastaran Behzadnia1, Monika M Golas2, Klaus Hartmuth1, Bjoern Sander2, Berthold Kastner1, Jochen Deckert1, Prakash Dube2, Cindy L Will1, Henning Urlaub3, Holger Stark2 and Reinhard Lührmann1
- Department of Cellular Biochemistry, MPI of Biophysical Chemistry, Göttingen, Germany
- 3D Electron Cryomicroscopy Group, MPI of Biophysical Chemistry, Göttingen, Germany
- Bioanalytical Mass Spectrometry Group, MPI of Biophysical Chemistry, Göttingen, Germany
Correspondence to:
Reinhard Lührmann, Department of Cellular Biochemistry, MPI for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany. Tel.: +49 551 2011407; Fax: +49 551 2011197; E-mail: reinhard.luehrmann@mpi-bpc.mpg.de
Holger Stark, 3D Electron Cryomicroscopy Group, MPI for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany. Tel.: +49 551 2011305; Fax: +49 551 2011197; E-mail: hstark1@gwdg.de
Received 13 July 2006; Accepted 2 February 2007
Abstract
Little is known about the higher-order structure of prespliceosomal A complexes, in which pairing of the pre-mRNA's splice sites occurs. Here, human A complexes were isolated under physiological conditions by double-affinity selection. Purified complexes contained stoichiometric amounts of U1, U2 and pre-mRNA, and crosslinking studies indicated that these form concomitant base pairing interactions with one another. A complexes contained nearly all U1 and U2 proteins plus 
50 non-snRNP proteins. Unexpectedly, proteins of the hPrp19/CDC5 complex were also detected, even when A complexes were formed in the absence of U4/U6 snRNPs, demonstrating that they associate independent of the tri-snRNP. Double-affinity purification yielded structurally homogeneous A complexes as evidenced by electron microscopy, and allowed for the first time the generation of a three-dimensional structure. A complexes possess an asymmetric shape (260 
200 195 Å) and contain a main body with various protruding elements, including a head-like domain and foot-like protrusions. Complexes isolated here are well suited for in vitro assembly studies to determine factor requirements for the A to B complex transition.
Keywords:
- electron microscopy,
- mass spectrometry,
- pre-mRNA splicing,
- Prp19/CDC5,
- spliceosomal A complex
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