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| Subject Categories: RNA | Structural Biology |
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| The EMBO Journal
(2007) 26, 1737–1748, doi:10.1038/sj.emboj.7601631 Published online 1 March 2007 |
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| Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes |
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| 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 |
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1 Department of Cellular Biochemistry, MPI of Biophysical Chemistry, Göttingen, Germany 2 3D Electron Cryomicroscopy Group, MPI of Biophysical Chemistry, Göttingen, Germany 3 Bioanalytical Mass Spectrometry Group, MPI of Biophysical Chemistry, Göttingen, Germany To whom correspondence should be addressed 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; Published online 1 March 2007. |
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| Abstract |
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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. |
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| Keywords: electron microscopy, mass spectrometry, pre-mRNA splicing, Prp19/CDC5, spliceosomal A complex |
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Top of page MORE ARTICLES LIKE THISThese links to content published by NPG are automatically generated NEWS AND VIEWSNature News and Views (07 Feb 1991) Nature News and Views (23 Sep 1993) RESEARCHThe EMBO Journal Article Isolation of an active step I spliceosome and composition of its RNP core Nature Article (17 Apr 2008) |
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