U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP.
Using an in vitro system in which a 5' splice site (5'SS) RNA oligo (AAG decreases GUAAGUAdT) is capable of inducing formation of U2/U4/U5/U6 snRNP complex we show that this oligo specifically binds to U4/U5/U6 snRNP and cross-links to U6 snRNA in the absence of U2 snRNP. Moreover, 5'SS RNA oligo bound to U4/U5/U6 snRNP is chased to U2/U4/U5/U6 snRNP complex upon addition of U2 snRNP. Recognition of the 5'SS by U4/U5/U6 snRNP correlates with the 5'SS consensus sequence. Unlike the interaction with U1 snRNP, this recognition depends largely on interactions other than RNA-RNA base pairing. Finally, the region of U6 snRNA required for this interaction with U4/U5/U6 snRNP is positioned upstream of stem I in the U4-U6 structure. We propose that the 5'SS-U4/U5/U6 snRNP complex is an intermediate in spliceosome assembly and that recognition of the 5'SS by U4/U5/U6 snRNP occurs after the 5'SS-U1 snRNA base pairing is disrupted but before the U4-U6 snRNA structure is destabilized.
Rockefeller University, New York, New York 10021.
B B
BB
Konforti
M M
MM
Konarska
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D012326:RNA Splicing / Q000235:genetics; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000235:genetics* / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics* / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism
D003432:Cross-Linking Reagents; D017412:Ribonucleoprotein, U1 Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D004247:DNA