OPR Protein-mediated Control and Adaption of Chloroplast Gene Expression in Chlamydomonas
Plant Genetics and Genomics
Plant Physiology
Final Report Abstract
In Chlamydomonas, numerous nucleus‐encoded members of the helical repeat protein family play prominent roles in the control of organellar gene expression. After their import into the chloroplast these factors act mostly at posttranscriptional steps to guarantee the expression of chloroplast encoded proteins by stabilizing specific mRNAs and/or by promoting their translation. To date, a predominant role of RNA binding Octotricopeptide Repeat (OPR) proteins as key players mediating these processes in Chlamydomonas is emerging. In this study, we present the characterization of the OPR protein MBC1, which is required for the stabilization of the psbC mRNA, encoding the photosystem (PS) II antenna protein CP43. By performing a primer‐based genome walking strategy, we identified a ~15 kb‐deletion in the mbc1 mutant strain retrieved from the Chlamydomonas library project. The deletion causes an almost complete loss of the MBC1 gene as well as the adjacent locus. The mutant exhibits a non‐photosynthetic phenotype with fluorescence induction kinetics and electrochromic shift analysis indicating the loss of PSII activity, while PSI and cytochrome b6f (Cytb6f) functionalities appeared unaffected. Molecular analysis of mbc1 revealed an abolished accumulation of psbC transcripts and the encoded CP43 protein. Complementation of the mutant with the MBC1 gene but not the adjacent locus fully restored psbC accumulation and photosynthetic performance. To further characterize the MBC1 protein, a knock‐in mutant expressing hemagglutinin (HA)‐tagged MBC1 proteins was generated via the CRISPR/Cas9 genome editing system. Size exclusion chromatography (SEC) of stromal proteins of this strain showed that MBC1‐HA is a component of a MDa‐sized RNase‐sensitive complex. To identify potential interaction partners of MBC1, a pull‐down of the tagged protein co‐precipitated several candidates, for one of which knock‐in and knock‐out strains were generated for further investigation. We additionally investigated the potential of MBC1’s putative minimal binding site within the 5’ UTR of the psbC mRNA to serve as minimal expression element for the stabilization and translation of unrelated mRNAs. The deep knowledge on distinct cis‐ and trans‐elements required for the expression of chloroplast mRNAs could improve the targeted manipulation of expression and biogenesis of photosynthetic complexes or the use of minimal intercistronic expression elements in synthetic operon‐derived mRNAs.
