Translation of partially overlapping psbD-psbC mRNAs in chloroplasts: the role of 5′-processing and translational coupling

3152–3158 Nucleic Acids Research, 2012, Vol. 40, No. 7 doi:10.1093/nar/gkr1185 Published online 8 December 2011 Translation of partially overlapping psbD-psbC mRNAs in chloroplasts: the role of 50-processing and translational coupling Yuka Adachi1, Hiroshi Kuroda1, Yasushi Yukawa1 and Masahiro Sugiura1,2,* 1 Graduate School of Natural Sciences, Nagoya City University, Yamanohata, Mizuho, Nagoya 467-8501 and Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan 2 Received October 17, 2011; Revised and Accepted November 14, 2011 ABSTRACT INTRODUCTION The chloroplast DNA in flowering plants is a circle 150 kb long, and includes approximately 80 different protein-encoding genes (1,2). Chloroplast gene expression is regulated in part at the transcriptional level (3,4), whereas posttranscriptional steps such as RNA editing, splicing, cleavage, trimming, translation and control of mRNA stability are more important for expression of most genes (5–11). Many chloroplast genes are clustered and are cotranscribed to polycistronic transcripts that are *To whom correspondence should be addressed. Tel: +81 52 872 6021; Fax: +81 52 872 6021; Email: ß The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The chloroplast psbD and psbC genes encode the D2 and CP43 proteins of the photosystem II complex, and they are generally cotranscribed. We report studies on the basic translation process of tobacco psbD-psbC mRNAs using an in vitro translation system from tobacco chloroplasts. The primary transcript has an unusually long 50 -UTR (905 nt). We show that it is translatable. Processing of the 50 -UTR greatly enhances the translation efficiency of the psbD cistron. A striking feature is that psbD and psbC cistrons overlap by 14 nt. Removal of the psbD 50 -UTR plus the start codon and introduction of a premature termination codon in the psbD cistron considerably reduce the translation efficiency of the downstream psbC cistron. These results indicate that translation of the psbC cistron depends largely on that of the upstream psbD cistron and thus shows translational coupling; however, a portion is independently translated. These observations, together with the presence of monocistronic psbC mRNAs, suggest that the psbD and psbC cistrons are translated via multiple processes to produce necessary amounts of D2 and CP43 proteins. then processed into mature RNAs (12–15). Some gene clusters consist of functionally unrelated genes; for example, the psaC-ndhD operon encodes a photosystem I subunit and an NADH dehydrogenase subunit (16). To express functionally unrelated cistrons from a polycistronic mRNA, regulation at posttranscriptional steps for each cistron would be required to supply proper amounts of products during development and under different environmental conditions. The photosystem II complex consists of approximately 20 different subunits, of which 14 are encoded in the chloroplast genome of flowering plants (17,18). Many of these genes are also clustered. One example is the clustered psbD gene for D2 protein and psbC gene for CP43 protein. A striking feature is that these two genes overlap by 14 nt in flowering plants (19–21), algae (22) and cyanobacteria (23). A notable exception is that these two genes are separated in Chlamydomonas reinhardtii (24). In flowering plants, the overlapping psbD and psbC genes (psbD/C) are cotranscribed with the downstream psbZ gene (formerly ORF62 or ycf9) to produce multiple overlapping transcripts (25,26). Transcription of monocot psbD/Cs starts from the preceding psbK and psbI gene cluster to give longer transcripts (27–29). The promoter for psbD/C is blue-light responsive (30,31) and is recognized by E. coli-type RNA polymerase with sigma factor 5 (32,33). However, little is known about the translation process of psbD/C mRNAs. In contrast, translation of separated psbD and psbC mRNAs in Chlamydomonas has been well studied using a biolistic chloroplast transformation system and heterologous reporter systems. Translation and stability of Chlamydomonas psbC and psbD mRNAs are controlled by interactions between 50 -UTRs and nucleus-encoded proteins (7–10). We previously reported that the tobacco monocistronic psbC mRNA is translatable (34). However, translation of the psbC cistron from the dicistronic psbD/C mRNA has not been investigated. In addition to being overlapping, the primary psbD/C transcript has an unusually long 50 -UTR. A portion of the primary transcript is processed Nucleic Acids Research, 2012, Vol. 40, No. 7 3153 MATERIALS AND METHODS The coding region for Cerulean (35) was prepared from the EGFP-coding region (36) and that for Citrine was obtained as described (36). Procedures for plasmid construction and primers are in Supplementary Figures S1 and S2. Necessary regions from psbD/C were amplified by PCR from tobacco chloroplast DNA (37) and PCR products were confirmed by DNA sequencing. Synthesis and purification of test mRNAs were as described (38). Test mRNA sequences are shown in Supplementary Figure S3. Isolation of intact tobacco chloroplasts and preparation of chloroplast extracts (S30 fractions) were as described (38). In vitro translation was performed at 28 C for 1 h as described (38). Protein products were separated by 12.5% native polyacrylamide gel electrophoresis (PAGE). Fluorescent signals were detected and quantified with a Typhoon 9400 system (GE Healthcare, Little Chalfont, UK). The 526SP and 555BP20 filters were used to detect Cerulean and Citrine bands when excited by 457-nm and 532-nm actinic light, respectively. RESULTS Measurement of translation from psbD and psbC cistrons Figure 1 shows a schematic of the tobacco chloroplast psbD-psbC-psbZ operon (17,37). These genes are cotranscribed to produce various mRNAs and the psbD/ C mRNA is one of the major transcripts accumulated in chloroplasts (25,26). In vitro capping assays have identified two transcription initiation sites: the main site is located 905-bp upstream from the psbD ATG start codon and the additional site is 230-bp upstream from the psbC GTG start codon (25,34). The primary psbD/C transcript has a 905-nt 50 -UTR with a processing site at position 132 relative to the A (+1) of the AUG start codon (25). To differentially monitor translation of the psbD and psbC cistrons in psbD/C mRNA, we utilized Cerulean (a cyan-fluorescent protein) and Citrine (a yellow-fluorescent protein). A large portion of the psbD cistron was replaced with the Cerulean-coding sequence (cerulean, 236 codons without start and stop codons), and that of the psbC cistron with a Citrine-coding frame (citrine, 237 codons without start codon) (Figure 2A). We (...truncated)