Isolation of an active step I spliceosome and composition of its RNP core.
Formation of catalytically active RNA structures within the spliceosome requires the assistance of proteins. However, little is known about the number and nature of proteins needed to establish and maintain the spliceosome's active site. Here we affinity-purified human spliceosomal C complexes and show that they catalyse exon ligation in the absence of added factors. Comparisons of the composition of the precatalytic versus the catalytic spliceosome revealed a marked exchange of proteins during the transition from the B to the C complex, with apparent stabilization of Prp19-CDC5 complex proteins and destabilization of SF3a/b proteins. Disruption of purified C complexes led to the isolation of a salt-stable ribonucleoprotein (RNP) core that contained both splicing intermediates and U2, U5 and U6 small nuclear RNA plus predominantly U5 and human Prp19-CDC5 proteins and Prp19-related factors. Our data provide insights into the spliceosome's catalytic RNP domain and indicate a central role for the aforementioned proteins in sustaining its catalytically active structure.
Bessonov
Sergey
S
Department of Cellular Biochemistry, Max Planck Institute of Biophysical Chemistry, D-37077 Göttingen, Germany.
Anokhina
Maria
M
Will
Cindy L
CL
Urlaub
Henning
H
Lührmann
Reinhard
R
D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D005091:Exons / Q000235:genetics; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012261:Ribonucleoproteins / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017381:Spliceosomes / Q000737:chemistry* / Q000235:genetics*
D046912:Multiprotein Complexes; D022821:RNA Splice Sites; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins