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 be lieved to require proteins, raising the possibility that splicing coul d be regulated; however, examples of such regulation have been lacking . The Chlamydomonas reinhardtii chloroplast psbA gene contains four la rge group I introns that self-splice efficiently in vitro, but only un der nonphysiological conditions. The psbA gene encodes the D1 protein of photosystem II, which is synthesized at very high rates in the ligh t in order to replace photodamaged protein. We show that psbA pre-mRNA s, containing one or more introns, accumulate in wild-type cells in th e dark, apparently due to rate-limited splicing. Analysis of the pre-R NAs indicates that splicing of the four introns does not follow a stri ct order. Exposure of cells to light induced rapid (15-20 min) decreas es in precursor levels of similar to 3-5-fold (depending on the intron ), which were accompanied by transient increases in free intron levels . Because light also stimulated psbA transcription similar to 2-fold o ver the same period, the data suggests that light increases the splici ng efficiency of psbA introns similar to 6-10-fold. Similar estimates of the extent of light stimulation were obtained by analyzing precurso r decay rates in the presence of actinomycin D. The effect of light is specific for psbA introns, because levels of unspliced 23S pre-RNA di d 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-chlorophenylhydr azone, 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 transforma nts of H13 that had been complemented with the tscA gene. These data s uggest strongly that light coordinately stimulates splicing of all fou r 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 discusse d.