INVOLVEMENT OF U1 SMALL NUCLEAR RIBONUCLEOPROTEINS (SNRNP) IN 5'-SPLICE SITE-U1 SNRNP INTERACTION

U1 small nuclear ribonucleoprotein (snRNP) is an important ribonucleop rotein involved early in the spliceosome formation to commit pre-mRNAs to the splicing pathway. We have determined the association and disso ciation kinetics of the 5' splice site-U1 snRNP interaction using puri fied U1 snRNP and a short RNA oligonucleotide comprising the 5' splice site (5'-SS) consensus sequence of pre-mRNAs (5'-SS RNA oligo). The a ssociation is rapid, does not require ATP, and is almost irreversible. Surprisingly, oligonucleotide-directed cleavage of the U1 small nucle ar RNA (snRNA) 5' end sequence with RNase H has no significant effect on the rate of association of the 5'-SS RNA oligo, but it does lead to rapid dissociation, This provides evidence that U1-specific snRNP pro teins are critical for the 5' splice site recognition while base pairi ng ensures the stability of the interaction. The recognition of the 5' splice site by U1 snRNP does not result from the individual action of one or more proteins but rather from their organization around U1 snR NA. A consequence of this organization is that the U1-C protein makes direct contacts with the site, as it becomes cross-linked to the RNA o ligo upon exposition of the reactions to shortwave UV light.