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...

Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start codon

“…Plasmids containing psbD 5′-UTR mutations were generated via mutagenesis PCR as described (23) with oligonucleotides 1963 and 1365 as well as oligonucleotides including the mutation, i.e. su2-a: 5′-gcaatgacaatttcgatcgg-3′; su2-b: 5′-ccgatcgaaattgtcattgc-3′; su4-a: 5′-gcaatgacaatggcgatcgg-3′; su4-b: 5′-ccgatcgccattgtcattgc-3′; su5-a: 5′-gagatacacacaatgacaat-3′; su5-b: 5′-attgtcattgtgtgtatctc-3′; revsu2-a: 5′-ggagatacacgaaatgacaa-3′; revsu2-b: 5′-ttgtcatttcgtgtatctcc-3′; revsu4-a: 5′-gagatacacgccatgacaat-3′; revsu4-b: 5′-attgtcatggcgtgtatctc-3′; revsu5-a: 5′-atgacaattgtgatcggtac-3′; revsu5-b: 5′-gtaccgatcacaattgtcat-3′; mutsu-a: 5′-ggagatacacgccatgacaa-3′; mutsu-b: 5′-ttgtcatggcgtgtatctcc-3′.…”

“…Initially, the cis -acting RNA targets were mapped by analyzing reporter gene constructs carrying mutated versions of distinct chloroplast 5′-UTRs (19–22) or by site-directed mutagenesis of the endogenous chloroplast gene regions (16,23,24). Alternatively, a chloroplast in vitro translation system from tobacco was used to define cis -acting translational elements within various plastid 5′-UTRs in higher plants (25,26).…”

“…This cis -acting element comprises a stretch of multiple U residues whose deletion in the mutant Δ U completely abolished the synthesis of the psbD gene product, i.e. the D2 protein of the photosystem II reaction centre (23), and, furthermore, led to the loss of RBP40-binding. A cis -acting suppressor of the Δ U mutation was isolated and shown to harbour a 5 bp duplication within the mutated region which partially restored both photosynthetic growth and RNA recognition by RBP40 (39).…”

Specific roles of 5' RNA secondary structures in stabilizing transcripts in chloroplasts

“…bacterial and organelle mRNAs, essential determinants of mRNA longevity seem to be located mostly in the 5′-UTR (6,7). Secondary structures at the 5′ ends of prokaryote-type mRNAs have been found to be important for mRNA stability, examples being stem–loops at the 5′ ends of mRNAs in Escherichia coli and Bacillus subtilis (8–10) and in the 5′-UTR of mRNAs in chloroplasts (11–13). Despite the importance of these 5′ secondary structures for mRNA longevity, their exact role in preventing rapid degradation of transcripts has been difficult to assess, even though it is often inferred that these structures function as binding sites for proteins and/or as structural elements that bar ribonucleases from attaching to and/or moving along the mRNA strand.…”

A spontaneous tRNA suppressor of a mutation in the Chlamydomonas reinhardtii nuclear MCD1 gene required for stability of the chloroplast petD mRNA

“…These data include the demonstration that Mbb1 and Nac2 are members of RNA-containing complexes (4,6), and the finding that cis element mutations in the 5′-UTRs of psbD (9) and petD (10), for example, can phenocopy the cognizant nuclear mutants.…”