Identification of RNA Editing Sites in Chloroplast Transcripts from the Maternal and Paternal Progenitors of Tobacco (Nicotiana tabacum): Comparative Analysis Shows the Involvement of Distinct Trans-Factors for ndhB Editing

Molecular Biology and Evolution, Jul 2003

RNA editing alters genomic nucleotide sequences at the transcript level. In higher plant chloroplasts, C-to-U conversion is known to occur at around 30 specific sites. The tobacco cultivar Nicotiana tabacum is an amphidiploid derived from ancestors of N. sylvestris (maternal) and N. tomentosiformis (paternal). The chloroplast genome of N. tabacum is believed to originate from an ancestor of N. sylvestris. To study the evolution of RNA editing in higher plant chloroplasts, editing sites in the two likely progenitors have first been identified based on those found in N. tabacum. Altogether 34, 33, and 32 editing sites have been found in the chloroplast transcripts from N. tabacum, N. sylvestris, and N. tomentosiformis, respectively. Thirty-one sites are conserved among the three species, whereas remarkable differences are observed in the editing of ndhB and ndhD transcripts. Sites 7 and 8 in ndhB mRNAs are separated only by five nt, and both are edited in N. tabacum and N. sylvestris. However, site 8 is not edited in N. tomentosiformis, indicating that distinct trans-factors are involved in the two editing events. The first site in ndhD mRNAs is edited to produce an AUG start codon in N. sylvestris as well as in N. tabacum but not in N. tomentosiformis, suggesting that a distinct mechanism operates for the translational initiation of N. tomentosiformis ndhD mRNAs. Four to six sites are edited partially in green leaves. Some of these sites may represent evolutionary intermediates in the process of losing editing events.

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Identification of RNA Editing Sites in Chloroplast Transcripts from the Maternal and Paternal Progenitors of Tobacco (Nicotiana tabacum): Comparative Analysis Shows the Involvement of Distinct Trans-Factors for ndhB Editing

Tadamasa Sasaki 0 Yasushi Yukawa 0 Tetsuya Miyamoto Junichi Obokata Masahiro Sugiura 0 0 Graduate School of Natural Sciences, Nagoya City University , Mizuho, Nagoya , Japan; and Center for Gene Research, Nagoya University , Nagoya , Japan RNA editing alters genomic nucleotide sequences at the transcript level. In higher plant chloroplasts, C-to-U conversion is known to occur at around 30 specific sites. The tobacco cultivar Nicotiana tabacum is an amphidiploid derived from ancestors of N. sylvestris (maternal) and N. tomentosiformis (paternal). The chloroplast genome of N. tabacum is believed to originate from an ancestor of N. sylvestris. To study the evolution of RNA editing in higher plant chloroplasts, editing sites in the two likely progenitors have first been identified based on those found in N. tabacum. Altogether 34, 33, and 32 editing sites have been found in the chloroplast transcripts from N. tabacum, N. sylvestris, and N. tomentosiformis, respectively. Thirty-one sites are conserved among the three species, whereas remarkable differences are observed in the editing of ndhB and ndhD transcripts. Sites 7 and 8 in ndhB mRNAs are separated only by five nt, and both are edited in N. tabacum and N. sylvestris. However, site 8 is not edited in N. tomentosiformis, indicating that distinct trans-factors are involved in the two editing events. The first site in ndhD mRNAs is edited to produce an AUG start codon in N. sylvestris as well as in N. tabacum but not in N. tomentosiformis, suggesting that a distinct mechanism operates for the translational initiation of N. tomentosiformis ndhD mRNAs. Four to six sites are edited partially in green leaves. Some of these sites may represent evolutionary intermediates in the process of losing editing events. Introduction The chloroplast is known to possess its own genome and gene expression system (Sugiura 1992). Many chloroplast genes in land plants are transcribed as polycistronic RNAs, which are then processed into mature RNA species via complex pathways including RNA editing and splicing (Sugiura, Hirose, and Sugita 1998; Rochaix 2001). RNA editing in chloroplasts was first reported in the maize rpl2 mRNA (Hoch et al. 1991), and a systematic search of chloroplast transcripts identified 27 editing sites in maize (Maier et al. 1995; Bock, Hermann, and Fuchs 1997), 26 editing sites in black pine (Wakasugi et al. 1996), 31 editing sites in tobacco (Hirose et al. 1999), and 21 editing sites in rice (Corneille, Lutz, and Maliga 2000). Editing in chloroplasts occurs generally in protein-coding regions and restored evolutionary conserved amino acid sequences (Maier et al. 1996). However, editing at the third position of a codon (silent editing) and editing in an untranslated region has also been reported (Hirose et al. 1996; Kudla and Bock 1999). In addition, extensive RNA editing, both C-to-U and U-to-C changes have been reported in the chloroplast of hornwort Anthoceros formosae (Yoshinaga et al. 1996). RNA editing has been found in chloroplast transcripts from all major lineages of land plants; however, neither frequency of editing nor the pattern of editing a specific transcript correlate with the phylogenic tree of the plant kingdom (Freyer, Kiefer-Meyer, and Kossel 1997). 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 Ko ssel. 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 cisacting 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 ele (...truncated)


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Tadamasa Sasaki, Yasushi Yukawa, Tetsuya Miyamoto, Junichi Obokata, Masahiro Sugiura. Identification of RNA Editing Sites in Chloroplast Transcripts from the Maternal and Paternal Progenitors of Tobacco (Nicotiana tabacum): Comparative Analysis Shows the Involvement of Distinct Trans-Factors for ndhB Editing, Molecular Biology and Evolution, 2003, pp. 1028-1035, 20/7, DOI: 10.1093/molbev/msg098