Pre-mRNA splicing requires dramatic RNA rearrangements hypothesized to be c
atalyzed by ATP-dependent RNA unwindases of the DExD/H box family. In a rea
rrangement critical for the fidelity of 5' splice site recognition, a base-
pairing interaction between the 5' splice site and U1 snRNA must be switche
d for a mutually exclusive interaction between the 5' splice site and U6 sn
RNA. By lengthening the U1:5' splice site duplex, we impeded this switch in
a temperature-dependent manner and prevented formation of the spliceosome'
s catalytic core. Using genetics, we identified the DExD/H box protein Prp2
8p as a potential mediator of the switch. In vitro, the switch requires bot
h Prp28p and ATP. We propose that Prp28p directs isomerization of RNA at th
e 5' splice site and promotes fidelity in splicing.