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. lepidoz- ioides 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 prerequi- site 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 A B 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.