Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes.
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.
Behzadnia
Nastaran
N
Department of Cellular Biochemistry, MPI of Biophysical Chemistry, Göttingen, Germany.
Golas
Monika M
MM
Hartmuth
Klaus
K
Sander
Bjoern
B
Kastner
Berthold
B
Deckert
Jochen
J
Dube
Prakash
P
Will
Cindy L
CL
Urlaub
Henning
H
Stark
Holger
H
Lührmann
Reinhard
R
D020029:Base Pairing / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D008958:Models, Molecular; D009841:Oligonucleotides; D011506:Proteins / Q000032:analysis*; D012322:RNA Precursors / Q000737:chemistry*; D017381:Spliceosomes / Q000737:chemistry*; D014031:Tobramycin
D009841:Oligonucleotides; D011506:Proteins; D012322:RNA Precursors; D014031:Tobramycin