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

Abstract

Using an in vitro system in which a 5′ splice site (5′SS) RNA oligo (AAG ↓ 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.