Highly conserved sequences at the 5' splice site and branch site of 
U12-dependent introns are important determinants for splicing by U12-dependent 
spliceosomes. This study investigates the in vivo splicing phenotypes of 
mutations in the branch site consensus sequence of the U12-dependent intron F 
from a human NOL1 (P120) minigene. Intron F contains a fully consensus branch 
site sequence (UUCCUUAAC). Mutations at each position were analyzed for their 
effects on U12-dependent splicing in vivo. Mutations at most positions resulted 
in a significant reduction of correct U12-dependent splicing. Defects observed 
included increased unspliced RNA levels, the activation of cryptic U2-dependent 
5' and 3' splice sites, and the activation of cryptic U12-dependent branch/3' 
splice sites. A strong correlation was observed between the predicted 
thermodynamic stability of the branch site: U12 snRNA interaction and correct 
U12-dependent splicing. The lack of a polypyrimidine tract between the branch 
site and 3' splice site of U12-dependent introns and the observed reliance on 
base-pairing interactions for correct U12-dependent splicing emphasize the 
importance of RNA/RNA interactions during U12-dependent intron recognition and 
proper splice site selection.