Site-specific cross-linking of mammalian U5 snRNP to the 5′ splice site before the first step of pre-mRNA splicing

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Abstract

We have used a site-specific cross-linking strategy to identify RNA and protein factors that interact with the 5′ splice site region during mammalian pre-mRNA splicing. Two different pre-mRNA substrates were synthesized with a single 32P-labeled 4-thiouridine residue 2 nucleotides upstream of the 5′ splice site. Selective photoactivation of the 4-thiouridine residue after incubation of either substrate under splicing conditions in HeLa nuclear extract resulted in cross-links to the U5 snRNA and the U5 snRNP protein p220. These ATP-dependent interactions occur before the first step of splicing. The U5 snRNA cross-links map to a phylogenetically invariant 9-nucleotide loop sequence and do not require Watson-Crick complementarity to the 5′ exon. Cross-links of this position in the pre-mRNA to U1, but not to U2, U4, or U6 snRNAs, were also observed. The kinetics of U1 and U5 cross-link formation are similar, both peaking well before reaction intermediates appear.

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Wyatt, J. R., Sontheimer, E. J., & Steitz, J. A. (1992). Site-specific cross-linking of mammalian U5 snRNP to the 5′ splice site before the first step of pre-mRNA splicing. Genes and Development, 6(12 PART B), 2542–2553. https://doi.org/10.1101/gad.6.12b.2542

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