RNA Editing in the Anticodon of Trna (CAA) Occurs Before Group I Intron

Plant Biology ISSN 1435-8603

RESEARCH PAPER RNA editing in the anticodon of tRNALeu (CAA) occurs before group I intron splicing in plastids of a moss Takakia lepidozioides S. Hatt. & Inoue Y. Miyata, C. Sugita, K. Maruyama & M. Sugita

Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan

Keywords ABSTRACT C-to-U editing; moss; plastid; RNA editing; Takakia lepidozioides; tRNALeu editing. RNA editing of cytidine (C) to uridine (U) transitions occurs in plastids and mitochondria of most land plants. In this study, we amplified and sequenced Leu Correspondence the group I intron-containing tRNA gene, trnL-CAA, from Takakia lepi- M. Sugita, Center for Gene Research, Nagoya dozioides, a moss. DNA sequence analysis revealed that the T. lepidozioides Leu University, Nagoya 464-8602, Japan. tRNA gene consisted of a 35-bp 5¢ exon, a 469-bp group I intron and a E-mail: [email protected] 50-bp 3¢ exon. The intron was inserted between the first and second position of the tRNALeu anticodon. In general, plastid tRNALeu genes with a group I Editor intron code for a TAA anticodon in most land plants. This strongly suggests M. Koornneef that the first nucleotide of the CAA anticodon could be edited in T. lepidozioides plastids. To investigate this possibility, we analysed cDNAs derived Received: 21 May 2007; Accepted: 11 July from the trnL-CAA transcripts. We demonstrated that the first nucleotide C 2007 of the anticodon was edited to create a canonical UAA anticodon in T. lepidozioides plastids. cDNA sequencing analyses of the spliced or doi:10.1111/j.1438-8677.2007.00027.x unspliced tRNALeu transcripts revealed that, while the spliced tRNA was completely edited, editing in the unspliced tRNAs were only partial. This is the first experimental evidence that the anticodon editing of tRNA occurs before RNA splicing in plastids. This suggests that this editing is a prerequisite to splicing of pre-tRNALeu.

2004). Of several hundreds sites, one editing site was INTRODUCTION identified in the tRNALeu (CAA) of A. capillus-veneris and RNA editing in plant mitochondria and plastids mostly in the tRNALys (UUC) of A. angustus. Both tRNAs are involves cytidine (C) to uridine (U) transitions, and encoded by an intron-containing gene. mainly affects mRNAs, thus providing the correct genetic Almost 200 sequences of the trnT-UGU and trnL-UAA information for the biosynthesis of proteins (Brennicke spacer and trnL 5¢ exon were sampled from various plant et al. 1999; Bock 2000). In seed plant mitochondria, sev- species, from bryophytes to seed plants (Quandt & Stech eral hundreds of editing sites are identified in most pro- 2004; Quandt et al. 2004). The nucleotide at the 3¢ end of tein-coding transcripts, and editing often allows the the 5¢ exon of trnL-UAA is a T in the plastid genomes maintenance of sequence conservation at the protein level from the most plant species, while it is a C in a moss (Shikanai 2006). Editing can also affect some of the plant Takakia lepidozioides, a gymnosperm Gingko biloba, and mitochondrial tRNAs (Fey et al. 2002). In angiosperm the leptosporangiate ferns sampled (except Osmunda plastids, 24 sites in sugarcane (Calsa et al. 2004) to 44 regalis) (Quandt & Stech 2004; Quandt et al. 2004). A RNA editing sites in moth orchid (Zeng et al. 2007) were T. lepidozioides sequence of part of a group I intron to identified only for protein-coding transcripts, and all are the trnL 3¢ exon was reported by Cox et al. (2004). The C-to-U changes. In contrast, 942 editing sites are known CAA anticodon of the intron-containing tRNALeu gene in the hornwort Anthoceros angustus (Kugita et al. 2003), was predicted from the two sequences (Cox et al. 2004; and 350 in the fern Adiantum capillus-veneris (Wolf et al. Quandt & Stech 2004). Therefore, the observed

250 Plant Biology 10 (2008) 250–255 ª 2008 German Botanical Society and The Royal Botanical Society of the Netherlands Miyata, Sugita, Maruyama & Sugita RNA editing in plastid tRNALeu anticodon substitution of T to C in the trnL 5¢ exon, located at the first position in the anticodon, might represent a prime candidate for possible tRNA anticodon editing. Alterna- tively, it is possible that the tRNALeu (CAA) gene may be a pseudogene in T. lepidozioides. Highly frequent RNA editing occurs in T. lepidozioides plastids (Sugita et al. 2006), and therefore also tRNALeu anticodon editing may be possible. In this study, we investigate whether C-to-U editing in the anticodon of tRNALeu (CAA) transcripts occurs in T. lepidozioides.We further investigate the editing–splicing status of the tRNA transcripts to clarify whether the editing occurs before or after splicing of the pre-tRNA. Fig. 1. Amplification of cDNAs derived from spliced or unspliced pre- tRNA. The schematic structure of pre-tRNALeu is shown at the top. Primers and the expected fragment sizes for PCR analysis are also MATERIALS AND METHODS shown. Amplification of a plastid DNA fragment Takakia lepidozioides S. Hatt. & Inoue was kindly provided 3 min, The amplified cDNA fragments were cloned into by Dr Masanobu Higuchi (National Science Museum, pGEM-T Easy. Tsukuba, Japan), who collected it in the field in Yu-shan, Taiwan. Total cellular DNA from T. lepidozioides DNA sequencing analysis was extracted as described previously (Sugita et al. 2006), and was used as a template for the polymerase DNA sequencing was performed using the DYEnamic ET chain reaction (PCR) with primers P1 (5¢-AACGATATTA Terminator Cycle Sequencing Kit and the ABI 3100 DNA CAACTTTTTCTCTC-3¢, AB301591) and P2 (5¢-TGTGTC sequencer. M13 universal primers, internal sequence AATGAAAAGAGATAGAAAC-3¢, AY312947). PCR was primers P3 (5¢-GGTCAAAAGGTTATTATTCGTTAG-3¢), carried out with an initial denaturation step at 95 C for and P4 (5¢-GTATGTTAGGTCATTCATTC-3¢) were used 3 min, followed by 30 cycles of denaturation at 95 C for for this study. Nucleotide sequences were analysed with 1 min, annealing at 55 C for 1 min, and extension at Genetyx-Mac 9.0 (Software Development Co., Japan) 72 C for 1 min; with a final extension at 72 C for 5 min. and were deposited in the DDBJ ⁄ EMBL ⁄ GenBank The amplified DNA fragment (743 bp) was cloned into databases, accession numbers AB304383 (T. lepidozioides pGEM-T Easy (Promega, Madison, WI, USA). trnL-CAA gene), AB299155 (spliced tRNALeu cDNA), AB299156 (edited pre-tRNALeu cDNA), and AB299157 (unedited pre-tRNALeu cDNA). Analysis of cDNA Total cellular RNA from T. lepidozioides was extracted RESULTS using Isogen (Nippon Gene, Toyama, Japan) and treated with RNase-free DNase I, as described previously (Sugita We amplified and sequenced the 743-bp plastid DNA et al. 2006). cDNAs were synthesised using random prim- fragment containing the tRNALeu gene, trnL-CAA, from ers as described previously (Miyata & Sugita 2004). For Takakia lepidozioides. DNA sequence analysis revealed amplification of cDNA derived from the spliced tRNALeu, that the T. lepidozioides tRNALeu gene consisted of a PCR was carried out using primers trnL-1 35-bp 5¢ exon, a 469-bp group I intron, and a 50-bp 3¢ (5¢-GGGGGTATGGCGGAATTGGTA-3¢) and trnL-3C exon. The intron was inserted between the first and (5¢-GGTTATTATTCGTTAGGATAGC-3¢), and an 85-bp second position of the tRNALeu anticodon (Fig. 2A). The DNA fragment was obtained (Fig. 1). Primer trnL-1 binds 5¢ exon sequence was identical to that reported by to the nucleotide positions 1 to 21 of the tRNA sequence Quandt & Stech (2004) and the 3¢ exon sequence was and primer trnL-3C binds to the positions 85–65 identical to that reported by Cox et al. (2004). The intron (Fig. 2A). For amplification of cDNA derived from unsp- sequence was different by two and three nucleotides to liced pre-tRNALeu, primers trnL-1 and trnL-2C those reported by Quandt & Stech (2004) and Cox et al. (5¢-TGGGGGTAGAGGGACTTGAAC-3¢) were used. The (2004), respectively. These differences may be due to primer trnL-2C hybridises to the intron of the cDNA single nucleotide polymorphisms derived from the differ- from unspliced pre-tRNA. An expected 195-bp DNA frag- ent geographic origins of T. lepidozioides plants (cf. Asia, ment was amplified. The PCR was carried out using Europe versus America). Ex Taq DNA polymerase with an initial denaturation step Group I intron-containing tRNALeu genes code for a at 94 C for 1 min, followed by 40 cycles of denaturation TAA anticodon in all land plants (Quandt & Stech 2004). at 94 C for 30 s, annealing at 50 C for 30 s, and exten- In contrast, the T. lepidozioides gene encodes a CAA sion at 72 C for 30 s; with a final extension at 72 C for anticodon (Fig. 2A). Hence, the C in the anticodon can

Plant Biology 10 (2008) 250–255 ª 2008 German Botanical Society and The Royal Botanical Society of the Netherlands 251 RNA editing in plastid tRNALeu anticodon Miyata, Sugita, Maruyama & Sugita

Fig. 2. A: Secondary structure deduced from the plastid DNA sequence of the T. lepidozioides tRNALeu (CAA) gene, trnL-CAA. The C (position 35) of the anticodon of tRNALeu is edited to U. An arrow indicates the insertion position of the group I intron. B: Predicted secondary structure of P1 of group I intron-containing pre-tRNALeu. The nucleotides of the editing site are indicated by bold letters. Edit converts a C–G pair (unedited) to the conserved U–G pair (edited) in the internal guide sequence (IGS), depicted in grey shading.

be edited in T. lepidozioides plastids. The expected editing synthesis by a secondary or tertiary structure of the group site could be base-paired with the internal guide sequence I intron. (IGS) in the conserved secondary structure P1 of the To investigate the editing–splicing status of transcripts group I intron (Fig. 2B). To investigate the occurrence of of the trnL-CAA gene, we amplified and sequenced the editing in the T. lepidozioides tRNALeu (CAA), we ampli- 195-bp cDNA fragment derived from unspliced forms of fied an 85-bp cDNA fragment derived from the spliced tRNALeu. Seven of 13 cDNAs were edited but six and mature tRNA molecule, and eight cDNA clones were remained unedited (Fig. 3). This result indicates that C to randomly selected and sequenced. As shown in Fig. 3, all U editing in the first nucleotide of the anticodon occurs of the eight clones were edited to T (U in RNA) at before splicing. position 35. PCR using a set of the same primers could amplify a 554-bp cDNA fragment containing the DISCUSSION group I intron of pre-tRNALeu. However, such a cDNA fragment was not amplified in this analysis. This may be In this study, we demonstrated that the first nucleotide C due to low amounts of the steady-state level of of the tRNALeu anticodon was edited to create a canonical unspliced pre-tRNA or a low efficiency of cDNA UAA anticodon in Takakia lepidozioides plastids. This is

252 Plant Biology 10 (2008) 250–255 ª 2008 German Botanical Society and The Royal Botanical Society of the Netherlands Miyata, Sugita, Maruyama & Sugita RNA editing in plastid tRNALeu anticodon

Fig. 3. RNA editing in spliced tRNA and unspliced tRNA. The editing sites are marked by an asterisk. The numbers of edited and ⁄ or unedited cDNAs are indicated. the third example of the occurrence of C-to-U editing in tRNA functioning only in the leptosporangiate fern a plastid tRNA anticodon. A partial C-to-U edit was Adiantum. To clarify the significance of time of editing, described in the anticodon of spliced tRNALeu (CAA) in before or after splicing, the editing–splicing status of plastids of the fern Adiantum (Wolf et al. 2004). From more leptosporangiate fern species and Ginkgo should be this description we can deduce that an RNA editing event evaluated. at this position occurs after group I intron splicing. In Cis-elements required for RNA editing in mRNAs were the hornwort Anthoceros, Kugita et al. (2003) found that identified within 20 nucleotides upstream and 10 nucleo- no editing was detected in the immature transcript of tides downstream from the edited position in land plant plastid trnK-UUC with a group II intron, however, cDNA plastids (Chaudhuri & Maliga 1996; Hirose & Sugiura synthesised within the tRNA revealed a C-to-U conver- 2001). A cis-element for editing of the C residue (at posi- sion at the 3¢ position of the anticodon UUC. This also tion 35) in the anticodon might be located within the supposes that tRNA splicing precedes RNA editing in the region of the 5¢-exon and its flanking sequence of the anticodon. In contrast, our data show that editing in the group I intron of T. lepidozioides tRNALeu. Therefore, a anticodon of tRNALeu occurrs before splicing, which is cis-element for editing may overlap with the sequence different from the reports from Adiantum tRNALeu and required for splicing of pre-tRNALeu. Editing in the Anthoceros tRNALys. T. lepidozioides plastid tRNALeu anticodon is a relatively Group I introns are characterised by five conserved ‘early’ RNA processing event at the exon site very close to core sequences (IGS, P, Q, R and S) and nine conserved the splice junction, as proposed in the case of plant mito- secondary structures (P1 to P9) (Burke et al. 1987). This chondrial pre-mRNAs (Sutton et al. 1991; Li-Pook-Than character also appears in the group I intron of T. lepidoz- et al. 2007). ioides tRNALeu (CAA) (Quandt & Stech 2004). The edit- In plant mitochondria, a C-to-U editing event corrects ing site is placed in the P1 duplex that is formed by a C:A mismatch into a U:A base pair in the acceptor interactions between the 5¢-exon and the IGS-containing stem of bean and potato mitochondrial tRNAPhe (GAA) sequences. Editing converts a C–G pair in IGS to the con- (Marećhal-Drouard et al. 1993) and in Oenothera served U–G pair (Fig. 2B), and thus would improve tRNAPhe and tRNACys (GCA) (Binder et al. 1994). In intron folding. The formation of this secondary structure larch (Larix leptoeuropaea) mitochondria, three C to U by editing could be required for splicing of the group I editing events restore U:A base pairs in the acceptor, D intron. If a G–C pair were edited to G–U, the intron– and anticodon stems, respectively, of tRNAHis (GUG) exon interaction would be weakened, and hence editing (Marećhal-Drouard et al. 1996). These editing events are might improve the efficiency of splicing. Thus, RNA edit- required for maturation and functioning of tRNA. Nei- ing is indispensable for both splicing and tRNA function- ther C-to-U editing in the tRNA anticodon nor in ing to provide the leucine tRNA in T. lepidozioides.In intron-containing tRNA genes has been identified in plant contrast, editing in the anticodon may be necessary for mitochondria so far.

Plant Biology 10 (2008) 250–255 ª 2008 German Botanical Society and The Royal Botanical Society of the Netherlands 253 RNA editing in plastid tRNALeu anticodon Miyata, Sugita, Maruyama & Sugita

Unlike T. lepidozioides and Adiantum tRNALeu (CAA), Calsa T., Jr, Carraro D.M., Benatti M.R., Barbosa A.C., the hornwort Anthoceros plastid tRNALeu has a UAA anti- Kitajima J.P., Carrer H. (2004) Structural features and tran- codon and does not require an RNA editing event script-editing analysis of sugarcane (Saccharum officinarum (Kugita et al. 2003). It is interesting to note that no gene L.) chloroplast genome. Current Genetics, 46, 366–373. Lys for tRNA is present in the Adiantum plastid genome Chaudhuri S., Maliga P. (1996) Sequences directing C to U (Wolf et al. 2003). There is no sequence data of a editing of the plastid psbL mRNA are located within a 22 Lys tRNA gene in the T. lepidozioides plastid genome. nucleotide segment spanning the editing site. The EMBO Therefore, we do not know whether RNA editing in the Journal, 15, 5052–5059. Lys tRNA anticodon found in Anthoceros is species-specific. Cox C.J., Goffinet B., Shaw A.J., Boles S.B. (2004) Phylogenetic For the other plastid tRNA genes, trnV-UAC, trnG-UCC, relationships among the mosses based on heterogeneous trnI-GAU and trnA-UGC that harbor an intron sequence Bayesian analysis of multiple genes from multiple genomic in the moss Physcomitrella patens (Sugiura et al. 2003) compartments. Systematic Botany, 29, 234–250. and in the liverwort Marchantia polymorpha (Ohyama Fey J., Weil J.H., Tomita K., Cosset A., Dietrich A., Small I., et al. 1986), RNA editing events have not yet been Marećhal-Drouard L. (2002) Role of editing in plant mito- predicted. Although these genes may be present in the T. lepidozioides plastid genome, if RNA editing occurs in chondrial transfer RNAs. Gene, 286, 21–24. these transcripts, it would be interesting to know how Hirose T., Sugiura M. (2001) Involvement of a site-specific editing and splicing events proceed in T. lepidozioides. trans-acting factor and a common RNA-binding protein in The Takakia genus comprises two species, T. lepidozio- the editing of chloroplast mRNAs: development of a chloro- ides and T. ceratophylla, and is of somewhat uncertain plast in vitro RNA editing system. The EMBO Journal, 20, placement. The exact placement of the genus Takakia 1144–1152. within mosses has been debated (Kenrick & Crane 1997; Kenrick P., Crane P.R. (1997) The origin and early evolution Newton et al. 2000). More recent molecular and morpho- of plants on land. Nature, 389, 33–39. logical data have solidified the placement of Takakia Kugita M., Yamamoto Y., Fujikawa T., Matsumoto T., among mosses (Cox et al. 2004; Shaw & Renzaglia 2004; Yoshinaga K. (2003) RNA editing in hornwort chloroplasts Qiu et al. 2006). RNA editing data may help to confirm makes more than half the genes functional. 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