Molecular architecture of the human Prp19/CDC5L complex.
Protein complexes containing Prp19 play a central role during catalytic activation of the spliceosome, and Prp19 and its related proteins are major components of the spliceosome's catalytic core RNP. To learn more about the spatial organization of the human Prp19 (hPrp19)/CDC5L complex, which is comprised of hPrp19, CDC5L, PRL1, AD002, SPF27, CTNNBL1, and HSP73, we purified native hPrp19/CDC5L complexes from HeLa cells stably expressing FLAG-tagged AD002 or SPF27. Stoichiometric analyses indicated that, like Saccharomyces cerevisiae NTC (nineteen complex), the human Prp19/CDC5L complex contains four copies of hPrp19. Salt treatment identified a stable core comprised of CDC5L, hPrp19, PRL1, and SPF27. Protein-protein interaction studies revealed that SPF27 directly interacts with each component of the hPrp19/CDC5L complex core and also elucidated several additional, previously unknown interactions between hPrp19/CDC5L complex components. Limited proteolysis of the hPrp19/CDC5L complex revealed a protease-resistant complex comprised of SPF27, the C terminus of CDC5L, and the N termini of PRL1 and hPrp19. Under the electron microscope, purified hPrp19/CDC5L complexes exhibit an elongated, asymmetric shape with a maximum dimension of approximately 20 nm. Our findings not only elucidate the molecular organization of the hPrp19/CDC5L complex but also provide insights into potential protein-protein interactions at the core of the catalytically active spliceosome.
Grote
Michael
M
Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
Wolf
Elmar
E
Will
Cindy L
CL
Lemm
Ira
I
Agafonov
Dmitry E
DE
Schomburg
Adrian
A
Fischle
Wolfgang
W
Urlaub
Henning
H
Lührmann
Reinhard
R
D026481:Blotting, Far-Western; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D012492:Salts / Q000494:pharmacology
C508357:CDC5L protein, human; D018797:Cell Cycle Proteins; D003432:Cross-Linking Reagents; D046912:Multiprotein Complexes; D016601:RNA-Binding Proteins; D012492:Salts