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)