Group II intron splicing in chloroplasts:: identification of mutations determining intron stability and fate of exon RNA

In order to investigate in vivo splicing of group ii introns in chloroplasts, we previously have integrated the mitochondrial intron rl1 from the green alga Scenedesmus obliquus into the Chlamydomonas chloroplast tscA gene, This construct allows a functional analysis of conserved intron sequences in vivo, since intron rl1 is correctly spliced in chloroplasts, Using site-directed mutagenesis, deletions of the conserved intron domains V and VI were performed. In another set of experiments, each possible substitution of the strictly conserved first intron nucleotide G1 was generated, as well as each possible single and double mutation of the tertiary base pairing gamma-gamma' involved in the formation of the intron's tertiary RNA structure, In most cases, the intron mutations showed the same effect on in vivo intron splicing efficiency as they did on the in vitro self-splicing reaction, since catalytic activity is provided by the intron RNA itself, In vivo, all mutations have additional effects on the chimeric tscA-rl1 RNA, most probably due to the role played by trans-acting factors in intron processing, Substitutions of the gamma-gamma' base pair lead to an accumulation of excised intron RNA, since intron stability is increased, In sharp contrast to autocatalytic splicing, all point mutations result in a complete loss of exon RNA, although the spliced intron accumulates to high levels. Intron degradation and exon ligation only occur in double mutants with restored base pairing between the gamma and gamma' sites. Therefore, we conclude that intron degradation, as well as the ligation of exon-exon molecules, depends on the tertiary intron structure. Furthermore, our data suggest that intron excision proceeds in vivo independent of ligation of exon-exon molecules.