Mutational analysis of the U12-dependent branch site consensus sequence.
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.
Brock
Jay E
JE
Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Dietrich
Rosemary C
RC
Padgett
Richard A
RA
D017398:Alternative Splicing; D001483:Base Sequence; D016384:Consensus Sequence; D006801:Humans; D007438:Introns; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism; D012359:tRNA Methyltransferases
D009687:Nuclear Proteins; D022821:RNA Splice Sites; D012342:RNA, Small Nuclear; C096986:U12 small nuclear RNA; C065028:NOP2 protein, human; D012359:tRNA Methyltransferases