Mutation at the chlamydomonas nuclear NAC2 locus specifically affects stability of the chloroplast psbD transcript encoding polypeptide D2 of PS II

Specific sequence elements in the 5′ untranslated regions of rbcL and atpB gene mRNAs stabilize transcripts in the chloroplast of Chlamydomonas reinhardtii

“…The sequences are predicted to participate in the formation of RNA secondary structures (Fig+ 1B)+ In Escherichia coli, the presence of a hairpin structure at the 59 ends of mRNAs has been found to be sufficient for extending the half-life of transcripts (Emory et al+, 1992;Arnold et al+, 1998), supposedly because binding or activity of RNase E, the endoribonuclease thought to do the initial nucleolytic attack on bacterial mRNAs, requires a short stretch of free 59 terminal nucleotides Mackie, 2000)+ However, relatively stable E. coli transcripts, having half-lives of 10-20 min, are still short-lived compared to chloroplast transcripts that have half-lives of several hours+ Considering the big difference in longevities of bacterial and chloroplast transcripts, and considering that only few of the transcript-destabilizing mutations introduced in this work into the rbc L and atpB 59 UTRs are predicted to destroy the presumptive original hairpin structures at the transcripts' 59 ends (as determined with the mfold program; Zuker et al+, 1999), 59 end structural features alone are unlikely to be sufficient to prolong half-lives of transcripts to several hours in the Chlamydomonas chloroplast+ There is considerable experimental evidence for an involvement of transcript-specific nuclear-encoded RNAbinding proteins that protect the RNAs against nucleolytic attack+ Analyses of several nuclear mutants of Chlamydomonas that do not accumulate specific chloroplast transcripts (Kuchka et al+, 1989;Sieburth et al+, 1991;Drapier et al+, 1992;Nickelsen, 2000) showed that nucleus-encoded protein factors are required for transcript stability in the chloroplast+ Recently, the nuclear Nac2 gene encoding a putative mRNA-stabilizing protein that is required for accumulation of transcripts of the Chlamydomonas chloroplast psbD gene has been FIGURE 4. Light/dark regulation of GUS transcript levels in Chlamydomonas chloroplast transformants carrying rbc L 59 end:GUS genes+ Transformants were grown in 12-h light/12-h dark cycles, total RNA was isolated at time points 11 h dark and 1 h light, and processed as described (legend to Fig+ 2; Materials and Methods)+ RNA gel blots were first hybridized to the atpB probe as a control and, after stripping of the membrane, to the GUS probe+ The increase upon illumination seen in abundance of transcripts of the endogenous atpB gene is due to increased transcription of the atpB gene in light and has been reported previously (Salvador et al+, 1993a)+ The membrane was exposed to X-ray film for 4 h for detection of atpB gene transcripts and for 24 h for detection of chimeric rbc L 59 end:GUS gene transcripts+ Numbers above the lanes denote nucleotide replacements in the rbc L 59 UTR sequence as depicted in Figures 1 and 2+ D: dark (also marked by a filled bar); L: light (also marked by an open bar)+ isolated by genomic complementation (Boudreau et al+, 2000)+ Putative cis-acting elements that are potential targets for RNA-stabilizing proteins have been localized in the 59 UTRs of chloroplast gene transcripts (Higgs et al+, 1999;Nickelsen et al+, 1999)+ A number of proteins were found in in vitro assays to bind to...…”

PHOTOSYSTEM II SUBUNIT R Is Required for Efficient Binding of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 to Photosystem II-Light-Harvesting Supercomplexes in Chlamydomonas reinhardtii

“…To address the question of whether PSBR is associated with PSII, we analyzed the presence of the protein in wild-type, PSII-deficient (nac2; Kuchka et al, 1989), and PSI-deficient (DpsaB; Redding et al, 1998) cells using immunoblotting after SDS-PAGE fractionation (Fig. 1).…”

Repression of Essential Chloroplast Genes Reveals New Signaling Pathways and Regulatory Feedback Loops inChlamydomonas

“…Transcripts for MetE had previously been shown to be repressed by the presence of exogenous vitamin B12 (Croft et al, 2005), so this was another possible regulatory element. The construct (detailed in Supplemental Data Set 1 online) was introduced into the nac2-26 mutant strain of C. reinhardtii, which is deficient in Nac2 expression and therefore unable to perform photosynthesis (Kuchka et al, 1989) (Figure 1A). The Nac2 protein is targeted to the chloroplast where it is specifically required for the expression of the chloroplast psbD gene encoding the PSII reaction center protein D2, which in turn is needed for photoautotrophic growth.…”