SPLICING OF A DIVERGENT SUBCLASS OF AT-AC INTRONS REQUIRES THE MAJOR SPLICEOSOMAL SNRNAS

AT-AC introns constitute a minor class of eukaryotic pre-mRNA introns, characterized by 5'-AT and AC-3' boundaries, in contrast to the 5'-GT and AG-3' boundaries of the much more prevalent conventional introns. In addition to the AT-AC borders, most known AT-AC introns have highl y conserved 5' splice site and branch site sequence elements of 7-8 nt . Intron 6 of the nucleolar P120 gene and intron 2 of the SCN4A voltag e-gated skeletal muscle sodium channel are AT-AC introns that have bee n shown recently to be processed via a unique splicing pathway involvi ng several minor U snRNAs. Interestingly, intron 21 of the same SCN4A gene and the corresponding intron 25 of the SCN5A cardiac muscle sodiu m channel gene also have 5'-AT and AC-3' boundaries, but they have div ergent 5' splice site and presumptive branch site sequences. Here, we report the accurate in vitro processing of these two divergent AT-AC i ntrons and show that they belong to a functionally distinct subclass o f AT-AC introns. Splicing of these introns does not require U12, U4ata c, and U6atac snRNAs, but instead requires the major spliceosomal snRN As U1, U2, U4, U5, and U6. Previous studies showed that G --> A mutati on at the first position and G --> C mutation at the last position of a conventional yeast or mammalian GT-AG intron suppress each other in vivo, suggesting that the first and last bases participate in an essen tial non-Watson-Crick interaction. Our results show that such introns, hereafter termed AT-AC II introns, occur naturally and are spliced by a mechanism distinct from that responsible for processing of the appa rently more common AT-AC I introns.