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.
Deshpande
N N
NN
Department of Botany, University of Texas at Austin, 78713, USA.
Bao
Y
Y
Herrin
D L
DL
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002736:Chloroplasts / Q000378:metabolism* / Q000528:radiation effects; D004579:Electron Transport; D018506:Gene Expression Regulation, Plant; D007438:Introns; D008027:Light; D010084:Oxidation-Reduction; D010775:Photic Stimulation; D010788:Photosynthesis / Q000235:genetics; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics*; D045331:Photosystem I Protein Complex; D045332:Photosystem II Protein Complex; D010940:Plant Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing / Q000528:radiation effects; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000528:radiation effects
D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D015800:Protozoan Proteins; D012322:RNA Precursors; D018749:RNA, Plant; D016053:RNA, Protozoan; C093455:tscA protein, Chlamydomonas