Role of the 3′ splice site in U12-dependent intron splicing

U12-dependent introns containing alterations of the 3 ' splice site AC dinucleotide or alterations in the spacing between the branch site and the 3 ' splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5 ' splice site AU dinucleotide, any nucleotide could serve as the 3 ' -terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3 ' splice site function. A branch site-to-3 ' splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3 ' splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3 ' splice site but that formation of the prespliceosomal complex A requires an active 3 ' splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3 ' splice site.