THE ROLE OF BRANCHPOINT-3' SPLICE-SITE SPACING AND INTERACTION BETWEEN INTRON TERMINAL NUCLEOTIDES IN 3' SPLICE-SITE SELECTION IN SACCHAROMYCES-CEREVISIAE

A conserved 3' splice site YAG is essential for the second step of pre -mRNA splicing but no trans-acting factor recognizing this sequence ha s been found. A direct, non-Watson-Crick interaction between the intro n terminal nucleotides was suggested to affect YAG selection. The mech anism of YAG recognition was proposed to involve 5' to 3' scanning ori ginating from the branchpoint or the polypyrimidine tract, We have con structed a yeast intron harbouring two closely spaced 3' splice sites, Preferential selection of a wildtype site over mutant ones indicated that the two sites are competing, For two identical sequences, the pro ximal site is selected, As previously observed, an A at the first intr on nucleotide spliced most efficiently with a 3' splice site UAC. In t his context, UAA or UAU were also more efficient 3' splice sites than UAG and competed more efficiently than the wild-type sequence with a 3 ' splice site UAC. We observed that a U at the first intron nucleotide is used for splicing in combination with 3' splice sites UAG, UAA or UAU, Our data indicate that the 3' splice site is not primarily select ed through an interaction with the first intron nucleotide. Selection of the 3' splice site depends critically on its distance from the bran chpoint but does not occur by a simple leaky scanning mechanism.