Pa. Maroney et al., Functional recognition of the 5 ' splice site by U4/U6.U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly, MOL CELL, 6(2), 2000, pp. 317-328
A sensitive assay based on competition between cis-and trans-splicing sugge
sted that factors in addition to U1 snRNP were important for early 5' splic
e site recognition. Cross-linking and physical protection experiments revea
led a functionally important interaction between U4/U6.U5 tri-snRNP and the
5' splice site, which unexpectedly was not dependent upon prior binding of
U2 snRNP to the branch point. The early 5' splice site/tri-snRNP interacti
on requires ATP, occurs in both nematode and HeLa cell extracts, and involv
es sequence-specific interactions between the highly conserved splicing fac
tor Prp8 and the 5' splice site. We propose that U1 and U5 snRNPs functiona
lly collaborate to recognize and define the 5' splice site prior to establi
shment of communication with the 3' splice site.