Mobile self-splicing group I introns from the psbA gene of Chlamydomonas reinhardtii: highly efficient homing of an exogenous intron containing its own promoter.

Introns 2 and 4 of the psbA gene of Chlamydomonas reinhardtii chloroplasts (Cr.psbA2 and Cr.psbA4, respectively) contain large free-standing open reading frames (ORFs). We used transformation of an intronless-psbA strain (IL) to test whether these introns undergo homing. Each intron, plus short exon sequences, was cloned into a chloroplast expression vector in both orientations and then cotransformed into IL along with a spectinomycin resistance marker (16S rrn). For Cr.psbA2, the sense construct gave nearly 100% cointegration of the intron whereas the antisense construct gave 0%, consistent with homing. For Cr.psbA4, however, both orientations produced highly efficient cointegration of the intron. Efficient cointegration of Cr.psbA4 also occurred when the intron was introduced as a restriction fragment lacking any known promoter. Deletion of most of the ORF, however, abolished cointegration of the intron, consistent with homing. The Cr.psbA4 constructs also contained a 3-(3,4-dichlorophenyl)-1,1-dimethylurea resistance marker in exon 5, which was always present when the intron integrated, thus demonstrating exon coconversion. Remarkably, primary selection for this marker gave >100-fold more transformants (>10,000/microgram of DNA) than did the spectinomycin resistance marker. A trans homing assay was developed for Cr.psbA4; the ORF-minus intron integrated when the ORF was cotransformed on a separate plasmid. This assay was used to identify an intronic region between bp -88 and -194 (relative to the ORF) that stimulated homing and contained a possible bacterial (-10, -35)-type promoter. Primer extension analysis detected a transcript that could originate from this promoter. Thus, this mobile, self-splicing intron also contains its own promoter for ORF expression. The implications of these results for horizontal intron transfer and organelle transformation are discussed.

The atpF group-II intron-containing gene from spinach chloroplasts is not spliced in transgenic Chlamydomonas chloroplasts.

In order to determine whether the group-II trans-splicing machinery of the chloroplast of Chlamydomonas reinhardtii can splice a heterologous group-II cis intron, the atpF gene of spinach was transferred into the chloroplast genome of C. reinhardtii using the atpX expression vector. The atpF gene contains a group-II intron which, like other higher plant chloroplast introns, does not self-splice in vitro. The chimeric transgene was expressed at high levels, based on the accumulation of the precursor; however, spliced products could not be detected by Northern blotting, or by RT-PCR coupled with Southern-blot hybridization of the amplified products with an exon-junction probe. These results indicate that the spinach atpF intron is not spliced in transgenic C. reinhardtii chloroplasts. Thus, splicing of chloroplast introns mediated by cellular factors may be species-specific; alternately, the group-II splicing machinery of C. reinhardtii is specific for trans spliced introns.

The catalytic group-I introns of the psbA gene of chlamydomonas reinhardtii : core structures, ORFs and evolutionary implications.

The sequences and predicted secondary structures of the four catalytic group-I introns in the psbA gene of Chlamydomonas reinhardtii, Cr.psbA-1-Cr.psbA-4, have been determined. Cr.psbA-1 and Cr.psbA-4 are subgroup-IA1 introns and have similar secondary structures, except at the 3' end where Cr.psbA-1 contains a large inverted-repeat domain. Cr.psbA-4 is closely related to intron 1 of the Chlamydomonas moewusii psbA gene, with which it shares the same location, high nucleotide identity in the core, and an identically placed ORF that shows 58% amino-acid identity. Cr.psbA-2 is a subgroup-IA3 intron, and shows similarities to the Chlamydomonas eugametos rRNA intron, Ce.LSU-1. Cr.psbA-3 is a subgroup-IA2 intron, and is remarkably similar to the T4 phage intron, sunY. Interestingly, a degenerate version of Cr.psbA-3 is located in the intergenic region between the chloroplast petA and petD genes. All four introns contain ORFs, which potentially code for basic proteins of 11-38 kDa. The ORFs in introns 2 and 3 contain variants of the GIY-YIG motif; however, the Cr.psbA-2 ORF is free-standing, whereas the Cr.psbA-3 ORF is contiguous and in-frame with the upstream exon. The Cr.psbA-4 ORF contains an H-N-H motif, and possibly a GIY-YIG motif. These data indicate that the C. reinhardtiipsbA introns have multiple origins, and illustrate some of the evolutionary DNA dynamics associated with group-I introns in Chlamydomonas.

Evidence for light/redox-regulated splicing of psbA pre-RNAs in Chlamydomonas chloroplasts.

Efficient splicing in vivo of most self-splicing group I introns is believed to require proteins, raising the possibility that splicing could be regulated; however, examples of such regulation have been lacking. The Chlamydomonas reinhardtii chloroplast psbA gene contains four large group I introns that self-splice efficiently in vitro, but only under nonphysiological conditions. The psbA gene encodes the D1 protein of photosystem II, which is synthesized at very high rates in the light in order to replace photodamaged protein. We show that psbA pre-mRNAs, containing one or more introns, accumulate in wild-type cells in the dark, apparently due to rate-limited splicing. Analysis of the pre-RNAs indicates that splicing of the four introns does not follow a strict order. Exposure of cells to light induced rapid (15-20 min) decreases in precursor levels of approximately 3-5-fold (depending on the intron), which were accompanied by transient increases in free intron levels. Because light also stimulated psbA transcription approximately 2-fold over the same period, the data suggests that light increases the splicing efficiency of psbA introns approximately 6-10-fold. Similar estimates of the extent of light stimulation were obtained by analyzing precursor decay rates in the presence of actinomycin D. The effect of light is specific for psbA introns, because levels of unspliced 23S pre-RNA did not decrease. The light-induced increase in psbA pre-RNA processing was abolished by inhibitors of photosynthetic electron transport, but not by the ATP synthesis inhibitor, carbonylcyanide m-chlorophenylhydrazone, which actually promoted pre-RNA processing in the dark. Finally, nonphotosynthetic mutants, including the tscA-lacking photosystem I mutant, H13, did not show evidence of light-stimulated RNA processing. However, the light response was restored in photosynthetic transformants of H13 that had been complemented with the tscA gene. These data suggest strongly that light coordinately stimulates splicing of all four psbA introns. Moreover, they demonstrate that this response to light is mediated by photosynthetic electron transport. The implications of these results for the regulation of psbA gene expression are discussed.