IN-VIVO COMMITMENT TO SPLICING IN YEAST INVOLVES THE NUCLEOTIDE UPSTREAM FROM THE BRANCH SITE CONSERVED SEQUENCE AND THE MUD2 PROTEIN

Pre-mRNA splicing is a stepwise nuclear process involving intron recog nition and the assembly of the spliceosome followed by intron excision . We previously developed a pre-mRNA export assay that allows the disc rimination between early steps of spliceosome formation and splicing p er se. Here we present evidence that these two assays detect different biochemical defects for point mutations. Mutations at the 5' splice s ite lead to pre-mRNA export, whereas 3' splice site mutations do not. Ii genetic screen applied to mutants in the branch site region shows t hat all positions in the conserved TACTAAC sequence are important for intron recognition. An exhaustive analysis of pre-mRNA export and spli cing defects of these mutants shows that the in vivo recognition of th e branch site region does not involve the base pairing of U2 snRNA wit h the pre-mRNA. In addition, the nucleotide preceding the conserved TA CTAAC sequence contributes to the recognition process. We show that a T residue at this position allows for optimal intron recognition and t hat in natural introns, this nucleotide is also used preferentially. M oreover, the Mud2 protein is involved in the recognition of this nucle otide, thus establishing a role for this factor in the in vivo splicin g pathway.