Site-specific cross-linking of mammalian U5 snRNP to the 5' splice site before the first step of pre-mRNA splicing.
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.
Wyatt
J R
JR
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, New Haven, Connecticut 06536-0812.
Sontheimer
E J
EJ
Steitz
J A
JA
D001483:Base Sequence; D017124:Conserved Sequence; D004247:DNA; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000235:genetics* / Q000378:metabolism; D017381:Spliceosomes
D012322:RNA Precursors; D017415:Ribonucleoprotein, U5 Small Nuclear; D004247:DNA