Principles of 3′ splice site selection and alternative splicing for an unusual group II intron from Bacillus anthracis

We investigated the self-splicing properties of two introns from the bacterium Bacillus anthracis. One intron (B.a.l1) splices poorly in vitro despite having typical structural motifs, while the second (B.a.l2) splices well while having apparently degenerated features. The spliced exons of B.a.l2 were sequenced, and splicing was found to occur at a 3' site shifted one nucleotide from the expected position, thus restoring missing gamma-gamma' and IBS3-EBS3 pairings, but leaving the two conserved exonic ORFs out of frame. Because of the unexpected splice site, the principles for 3' intron definition were examined, which showed that the 3' splice site is flexible but contingent on gamma-gamma' and IBS3-ERS3 pairings, and can be as far away as four nucleotides from the wild-type site. Surprisingly, alternative splicing occurs at position +4 for wild-type B.a.l2 intron, both in vitro and in vivo, and the alternative event fuses the two conserved exon ORFs, presumably leading to translation of the downstream ORF. The finding suggests that the structural irregularities of B.a.l2 may be an adaptation to facilitate gene expression in vivo.