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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.

U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP.