Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes
- 1 March 2007
- journal article
- research article
- Published by Springer Science and Business Media LLC in The EMBO Journal
- Vol. 26 (6), 1737-1748
- https://doi.org/10.1038/sj.emboj.7601631
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
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