* Graduate School of Natural Sciences, Nagoya City University, Mizuho, Nagoya, Japan
Center for Gene Research, Nagoya University, Nagoya, Japan
Correspondence: E-mail: sugiura{at}nsc.nagoya-cu.ac.jp.
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Abstract |
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Key Words: chloroplast RNA editing trans-factor transcript tobacco
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Introduction |
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A key question in chloroplast editing is how specific C residues are recognized precisely from all other C residues in transcripts. Using transgenic approaches in tobacco chloroplasts, cis-acting elements have been analyzed for psbL mRNAs (Chaudhuri, Carrer, and Maliga 1995; Chaudhuri and Maliga 1996), for ndhB mRNAs (sites 4 and 5) (Bock, Hermann, and Kössel. 1996; Bock, Hermann, and Fuchs 1997; Hermann and Bock 1999), and for ndhF and rpoB (site 2) mRNAs (Reed, Lyi, and Hanson 2001). These studies commonly showed that cis-acting elements reside in upstream regions of the editing sites. Furthermore, chloroplast transplastomic experiments suggested the involvement of trans-acting factors in editing (Chaudhuri, Carrer, and Maliga 1995; Chaudhuri and Maliga 1996; Bock and Koop 1997; Reed and Hanson 1997; Reed, Lyi, and Hanson 2001; Schmitz-Linneweber et al. 2001). These in vivo analyses show that at least some trans factors appear to be site specific and of extraplastidic origin. Recently, an in vitro RNA editing system from tobacco chloroplasts was developed in our laboratory to dissect biochemical processes of editing reactions in chloroplasts (Hirose and Sugiura 2001). Using this system, a tobacco chloroplast protein of 25 kd was found to bind specifically to the cis-acting element of psbL mRNA. This result provided the evidence that the protein, but not RNA, is the trans-acting factor that is likely to recognize the editing site of psbL mRNAs. An improved method was then reported for preparing chloroplast extracts supporting accurate RNA editing reactions in vitro not only from tobacco but also from pea (Miyamoto, Obokata, and Sugiura 2002). Using this improved system, we defined cis elements of psbE and petB mRNAs and detected trans factors that specifically bind to these elements, 56-kd and 70-kd proteins for psbE and petB mRNAs, respectively.
In the case of tobacco chloroplasts, the genome sequence has been completely determined (Shinozaki et al. 1986), the gene organization has been updated (Wakasugi et al. 1998; Wakasugi, Tsudzuki, and Sugiura 2001), and a systematic search for editing sites in the transcripts has been made (Hirose et al. 1999). In addition, both chloroplast transformation techniques (in vivo) (Svab and Maliga 1993) and chloroplast RNA editing system (in vitro) (Hirose and Sugiura 2001) are available only for tobacco. Therefore, tobacco is the organism of choice for analyzing detailed mechanisms of RNA editing in chloroplasts. The tobacco cultivar Nicotiana tabacum is a natural amphidiploid derived from two progenitors, and ancestors of N. sylvestris and N. tomentosiformis were the likely progenitors of N. tabacum (Smith 1974; Kenton et al. 1993). The chloroplast genome of N. tabacum is believed to have originated from N. sylvestris (Olmstead and Palmer 1991). Hence, these Nicotiana species offer a significant advantage for evolutional studies of RNA editing in higher plant chloroplasts. Recently, Schmitz-Linneweber et al. (2001) reported an interesting observation that N. tabacum lost an editing site but still possesses its trans factor, which probably originated from a progenitor of N. tomentosiformis.
Here we report the editing pattern of both N. sylvestris and N. tomentosiformis chloroplasts. Comparative analysis with editing sites in N. tabacum shows the opposite case as above, namely the presence of a C residue (to be edited in N. tabacum and N. sylvestris) but no corresponding editing activity in N. tomentosiformis. This analysis also indicates the involvement of distinct trans factors for two adjacent sites.
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Materials and Methods |
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Results and Discussion |
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As shown in figure 1A, ndhB sites 7 and 8 in N. tabacum are separated only by 5 nt (one alanine codon, GCU) and both editing events cause an amino acid substitution from serine (UCA) to leucine (UUA). This is also the case for N. sylvestris, whereas editing was not observed in N. tomentosiformis for the C residue corresponding to site 8 of N. tabacum (fig. 1B). cDNA sequencing was repeated three times with different RNA preparations, and the same results were obtained. Therefore, we concluded that N. tomentosiformis lacks editing activity for site 8 despite conserved sequences around this position among the three Nicotiana species. On the other hand, another pair of ndhB sites 5 and 6, separated only by 8 nt, are edited in N. tomentosiformis as in N. tabacum and N. sylvestris (fig. 1B). These results indicate that site-recognition factors for sites 7 and 8 are different, because editing should occur in both sites if a single factor recognizes both C residues to be edited. This is consistent with the observation on transplastomic tobacco lines that the ndhB sites 7 and 8 are edited independently (Bock, Hermann, and Fuchs 1997).
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Silent Editing in atpA Transcripts
The atpA gene encodes the -subunit of ATP synthase complex and is located at the last gene in the rps2-atpIHFA operon (Wakasugi et al. 1998). Transcription of this operon in N. tabacum starts from at least four sites, and resulting polycistronic mRNAs are processed from at least four sites to produce a dicistromic atpF-atpA mRNA and other mRNAs (Miyagi et al. 1998). As shown in figure 3A, two editing sites were found in two successive codons of N. tabacum atpA transcripts (Hirose et al. 1996). The first C-to-U conversion caused proline (CCC) to leucine (CUC) substitution, whereas the second editing took place partially at the third position of the serine codon (UCC to UCU), leading to no amino acid change (silent editing). In the case of N. sylvestris and N. tomentosiformis atpA mRNAs, editing was detected in the first codon but not in the second position (fig. 3B), as was found in pea atpA mRNAs (Hirose et al. 1996). Therefore, silent editing at the serine codon seems to be unique to N. tabacum.
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We used transcripts from young green leaves to identify editing sites in the Nicotiana species. Most identified sites are fully edited, whereas a limited number of identified sites are edited partially (table 2). Partial editing was confirmed by three independent cDNA sequencing from different RNA preparations. The extent of editing is dependent on the site. In N. tabacum, partial editing was reported in atpA site 2 (Hirose et al. 1996), ndhD site 1 (Hirose and Sugiura 1997) and rpoA (Hirose et al. 1999). An additional site, ndhD site 3, was found from our own analysis to be partially edited (fig. 4). Altogether four sites are partially edited in N. tabacum green leaves.
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Editing of ndhB sites 3 and 4 and rpoC1 occurs fully in N. tabacum (Hirose et al. 1999) but not in the other species. This phenomenon could be explained by doubling nuclear genes encoding editing factors. Recently, it was reported that editing of the two ndhB sites in N. tabacum is temperature sensitive (Karcher and Bock 2002). Therefore, interaction of cis elements with editing machineries for these editing sites may be more fragile when compared with other sites. The corresponding sites in maize plastids were poorly edited in roots and calli (Peeters and Hanson 2002), suggesting low expression of the trans factors in nonphotosynthetic cells. However, the opposite case was observed for rpoA editing; partial in the tetraploid but full in the two diploid species. This may be due to interference in N. tabacum between the expression of nuclear genes encoding the editing factors or between these factors derived from the two progenitors. Partial editing implies the presence of two or more different mRNA species from single genes, which potentially leads to the microheterogeneity of relevant protein products. If this is not the case, mRNA surveillance mechanisms should operate in chloroplasts. In the case of an editing event creating AUG start codons, unedited mRNAs are most likely nonfunctional.
Conclusion
Recent findings show that an editing activity can be present despite the absence of the target site; N. tabacum is capable of editing the exogenous ndhA site 2 even though its plastid genome lacks this site (Schmitz-Linneweber et al. 2001). The nucleus of N. tomentosiformis is suggested to be the donor of the corresponding trans factor. Here we present evidence to the contrary; N. tabacum and N. sylvestris have ndhB site 8 and ndhD site 1 and their cognate editing activities, whereas no editing activity for these sites in N. tomentosiformis was detected even though these positions hold C residues in their genome. Therefore, these editing factors are thought to originate from N. sylvestris. As for the ndhB site 8, the ndhB protein may be functional with either serine or leucine at this position. If this is the case, editing of this site is dispensable and N. tomentosiformis lost its editing activity. This suggests that conserved amino acid residues are not always essential for protein function. Comparison of editing patterns among N. tabacum (amphidiploid) and its progeny representatives, N. sylvestris and N. tomentosiformis, will provide clues for better understanding of the evolution of editing events in plastids.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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