In fission yeast, Prp4 kinase activity is required for recognition of introns with weak splice sites. In this context, we analyzed the consequences of mutations within the branch sequence. We found that only changes in the nucleotide transiently interacting with a pseudouridine in snRNA U2 and one double mutation in the branch sequence lead to the requirement of Prp4 kinase activity. The results presented here and previously let us propose that Prp4 kinase activity is part of a safeguarding process that facilitates formation of transient RNA duplexes between pre-mRNAs and snRNAs to form the pre-spliceosome.