Competing 5' splice sites of SRC1/HEH1 are marked for alternative splicing by U5 and U6 snRNAs during the B to Bact transition of the spliceosome
Alternative splicing of precursor-messenger RNA (pre-mRNA) bearing introns with competing and overlapping 5 splice sites (5SS) produces more mRNAs. We investigated the mechanism of this form of alternative splicing by monitoring RNA and protein products of the yeast SRC1/HEH1 gene. Its alternative splicing requires a sixteen-nucleotide pre-mRNA segment spanning the two 5SS. The nucleotides are decoded by U5 and U6 small nuclear RNAs (snRNA), further supported by proteins of the spliceosomal B and Bact complexes. Specific variants of the pre-mRNA segment with recalibrated binding to U5 and U6 snRNAs allowed alternative splicing independently of the trans-acting proteins. Conversely, snRNA variants specified for modified 5SS selection suppressed the splicing defects in the B complex mutants. Further data suggest that alternative splicing from HEH1-type introns containing overlapping and competing 5SS relies on pre-mRNA-snRNA interactions during B to Bact transitions of the spliceosome, supported by proteins that stabilize low-fidelity, high-efficiency conformations.