Nucleic Acids Research, Vol 27, Issue 11 2345-2353, Copyright © 1999 by Oxford University Press
V Hollander and U Kuck
In order to investigate in vivo splicing of group II introns in
chloroplasts, we previously have integrated the mitochondrial intron rI1
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 rI1 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 -rI1 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.
ARTICLES
Group II intron splicing in chloroplasts: identificationof mutations determining intron stability and fate of exon RNA
Lehrstuhl fur Allgemeine Botanik, Fakultat fur Biologie, Ruhr- Universitat Bochum, D-44780 Bochum, Germany.
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