A functional association between the 5' and 3' splice site is established in the earliest prespliceosome complex (E) in mammals.

The earliest detectable mammalian prespliceosome complex (E) contains the non-snRNP splicing factor U2AF, U1 snRNP, and several spliceosome-associated proteins (SAPs). We show that specific complexes, designated E3' and E5', assemble independently on RNAs containing only a 3' or 5' splice site, respectively. U2AF is enriched in E3', whereas U1 snRNP is enriched in E5'. Using a highly sensitive substrate-competition assay, we show that both the 5' splice site and the pyrimidine tract at the 3' splice site are required for efficient E complex assembly on intact pre-mRNA. We conclude that the 5' and 3' splice sites are associated functionally as early as E complex by either direct or indirect interactions between U1 snRNP and U2AF. Our observations predict that E complex assembly is a major control point for establishing splice site selection in both constitutively and alternatively spliced pre-mRNAs.