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 which small nuclear ribonucleoprotein particles (snRNPs) and some non-snRNP proteins recognize the splice site sequences and undergo various conformational rearrangements. A number of important intermolecular RNA-RNA duplexes are formed transiently during the process of splice site recognition. Various steps in the assembly pathway are dependent upon ATP hydrolysis, either for protein phosphorylation or for the activity of helicases, which may modulate the RNA structures. Major efforts have been made to identify proteins that interact with specific regions of 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-paired RNA. Here we have used the complementary approach of methylene blue-mediated photo-cross-linking to detect specifically proteins that interact with 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 site. 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 splicing.