DETECTION OF A NOVEL ATP-DEPENDENT CROSS-LINKED PROTEIN AT THE 5'-SPLICE SITE-U1 SMALL NUCLEAR-RNA DUPLEX BY METHYLENE BLUE-MEDIATED PHOTO-CROSS-LINKING

Assembly of spliceosomes involves a number of sequential steps in whic h small nuclear ribonucleoprotein particles (snRNPs) and some non-snRN P proteins recognize the splice site sequences and undergo various con formational rearrangements. A number of important intermolecular RNA-R NA duplexes are formed transiently during the process of splice site r ecognition. Various steps in the assembly pathway are dependent upon A TP hydrolysis, either for protein phosphorylation or for the activity of helicases, which may modulate the RNA structures. Major efforts hav e been made to identify proteins that interact with specific regions o f the pre-mRNA during the stages of spliceosome assembly and catalysis by site-specific UV cross-linking. However, UV cross-linking is often inefficient for the detection of proteins that interact with base-pai red RNA. Here we have used the complementary approach of methylene blu e-mediated photo-cross-linking to detect specifically proteins that in teract,vith the duplexes formed between pre-mRNA and small nuclear RNA (snRNA), We have detected a novel cross-link between a 65-kDa protein (p65) and the 5' splice site. A range of data suggest that p65 cross- links to the transient duplex formed by U1 snRNA and the 5' splice sit e. Moreover, although p65 cross linking requires only a 5' splice site within the pre-mRNA, it also requires ATP hydrolysis, suggesting that its detection reflects a very early ATP-dependent event during splici ng.