Splicing factor Prp8 governs U4/U6 RNA unwinding during activation of the spliceosome

The pre-mRNA 5' splice site is recognized by the ACAGA box of U6 spliceosom al RNA prior to catalysis of splicing. We previously identified a mutant U4 spliceosomal RNA, U4-cs1, that masks the ACAGA box in the U4/U6 complex, t hus conferring a cold-sensitive splicing phenotype in vivo. Here, we show t hat U4-cs1 blocks in vitro splicing in a temperature-dependent, reversible manner. Analysis of splicing complexes that accumulate at low temperature s hows that U4-cs1 prevents U4/U6 unwinding, an essential step in spliceosome activation. A novel mutation in the evolutionarily conserved U5 snRNP prot ein Prp8 suppresses the U4-cs1 growth defect. We propose that wild-type Prp 8 triggers unwinding of U4 and U6 RNAs only after structurally correct reco gnition of the 5' splice site by the U6 ACAGA box and that the mutation (pr p8-201) relaxes control of unwinding.