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- Title
Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes.
- Authors
Behzadnia, Nastaran; Golas, Monika M; Hartmuth, Klaus; Sander, Bjoern; Kastner, Berthold; Deckert, Jochen; Dube, Prakash; Will, Cindy L; Urlaub, Henning; Stark, Holger; Lührmann, Reinhard
- 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 approximately 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 (approximately 260 x 200 x 195 angstroms) 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.
- Publication
The EMBO journal, 2007, Vol 26, Issue 6, p1737
- ISSN
0261-4189
- Publication type
Journal Article
- DOI
10.1038/sj.emboj.7601631