Identification of cis-Acting RNA Leader Elements Required for Chloroplast psbD Gene Expression in Chlamydomonas

Abstract

The psbD mRNA of Chlamydomonas reinhardtii is one of the most abundant chloroplast transcripts and encodes the photosystem II reaction center polypeptide D2. This RNA exists in two forms with 5 Ј untranslated regions of 74 and 47 nucleotides. The shorter form, which is associated with polysomes, is likely to result from processing of the larger RNA. Using site-directed mutagenesis and biolistic transformation, we have identified two major RNA stability determinants within the first 12 nucleotides at the 5 Ј end and near position Ϫ 30 relative to the AUG initiation codon of psbD. Insertion of a polyguanosine tract at position Ϫ 60 did not appreciably interfere with translation of psbD mRNA. The same poly(G) insertion in the nac2-26 mutant, which is known to be deficient in psbD mRNA accumulation, stabilized the psbD RNA. However, the shorter psbD RNA did not accumulate, and the other psbD RNAs were not translated. Two other elements were found to affect translation but not RNA stability. The first comprises a highly U-rich sequence (positions Ϫ 20 to Ϫ 15), and the second, called PRB1 (positions Ϫ 14 to Ϫ 11), is complementary to the 3 Ј end of the 16S rRNA. Changing the PRB1 sequence from GGAG to AAAG had no detectable effect on psbD mRNA translation. However, changing this sequence to CCUC led to a fourfold diminished rate of D2 synthesis and accumulation. When the psbD initiation codon was changed to AUA or AUU, D2 synthesis was no longer detected, and psbD RNA accumulated to wild-type levels. The singular organization of the psbD 5 Ј untranslated region could play an important role in the control of initiation of psbD mRNA translation. INTRODUCTIONChloroplast gene expression has been shown to be regulated at various levels, including transcription and several post-transcriptional steps, such as RNA stabilization, RNA processing, and RNA splicing and translation. Genetic analysis of the unicellular green alga Chlamydomonas reinhardtii and higher plants suggests that these processes are controlled in large part by nucleus-encoded factors. These factors are synthesized in the cytosol and subsequently imported into the chloroplast, where they interact with their cognate target sites on either chloroplast RNAs or proteins to fulfill their function (Rochaix, 1996;Sugita and Sugiura, 1996; Goldschmidt-Clermont, 1998).The availability of a biolistic chloroplast transformation system (Boynton et al., 1988) has allowed us to analyze sitedirected chloroplast mutations and to use heterologous reporter systems. This has led to the identification of essential cis -acting RNA elements on chloroplast mRNAs that are involved in the regulation of gene expression. As an example, the analysis of chloroplast transformants revealed the importance of 5 Ј untranslated regions (UTRs) for the regulation of translation of the psbC (Zerges and Rochaix, 1994;Zerges et al., 1997), psbA (Mayfield et al., 1994), petD (Sakamoto et al., 1994), and psaB (Stampacchia et al., 1997) mRNAs in Chlamydomonas. They encode subunits of photosynthetic c...

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

“…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 the 59 UTRs of Chlamydomonas chloroplast transcripts (Hauser et al+, 1996) and, in spinach, to the 59 UTRs of transcripts of genes that code for subunits of the ATP synthase complex (Hotchkiss & Hollingsworth, 1999)+ Although the identities and functions of these proteins have not been examined, it is possible that some of them are nucleus-encoded factors involved in mRNA stabilization+…”

Efficient Assembly of Photosystem II in Chlamydomonas reinhardtii Requires Alb3.1p, a Homolog of Arabidopsis ALBINO3

“…In vivo labeling of C. reinhardtii with [ 35 S] was performed as described previously (Nickelsen et al, 1999). For the pulse/chase analysis, the medium containing [ 35 S] was replaced after 15 min by nonradioactive medium, and the incubation was prolonged for 60 min.…”

Chloroplast RNA-binding proteins

“…The same applies to 5¢UTRs of psbD, petD and psbB transcripts in C. reinhardtii, which were shown to contain the target sites for nucleus-encoded factors involved in the gene-specific stabilization of the corresponding mRNAs (Nickelsen et al 1994;Drager et al 1998;Vaistij et al 2000a). For psbD and petD RNAs, site-directed mutagenesis enabled the precise localization of distinct RNA elements mediating RNA stabilization (Higgs et al 1999;Nickelsen et al 1999).…”

“…Analysis of the photosynthetic mutant nac2 revealed that the stability of the psbD mRNA depends on a nucleus-encoded tetratricopeptide repeats protein, which is part of a highmolecular-weight complex mediating its function via the psbD 5¢UTR (Boudreau et al 2000). However, a thorough site-directed mutagenesis of the psbD 5¢region after biolistic transformation of chloroplasts from wild-type cells identified several essential cis-acting elements required for either stabilization or translation of the psbD message (Nickelsen et al 1999). Concomitant in vitro RNA binding assays with the mutant psbD 5¢ versions then demonstrated that the deletion of an U-rich element leading to a defect in psbD mRNA translation in vivo resulted in the inability of binding a protein of 40 kDa (RBP40) to the 5¢UTR in vitro.…”

“…1. Indirect evidence strongly suggests that, after transcription, the Nac2 complex interacts with a RNA element within the psbD 5¢UTR, called PRB2 (Nickelsen et al 1999). This leads to the protection of the RNA against exonucleolytic degradation from the 5¢ end of the message.…”