Extensive RNA Editing of U to C in Addition to C to U Substitution in the rbcL Transcripts of Hornwort Chloroplasts and the Origin of RNA Editing in Green Plants

Koichi Yoshinaga 0 2 Hiroe Iinuma 0 2 Takehiro Masuzawa 0 2 Kunihiko Uedal 0 1 2 0 Kanazawa 920-11, Japan 1 Faculty of Science, Kanazawa University 2 Faculty of Science, Shizuoka University , Shizuoka 422, Japan We cloned and sequenced a portion of chloroplast DNA from the hornwort Anthoceros formosae. A nucleotide sequence of 7556 bp contained structures similar to those of ndhK, ndhC, trnV, trnM, atpE, atpB, rbcL, trnR and accD. The arrangement of these was the same as that of other chloroplast DNA. However, two nonsense codons were located within the putative coding region of rbcL, although they were used as putative termination codons of the genes. RNA was extensively edited in the transcripts of rbcL when cDNA sequences were analyzed. The unusual nonsense codons of TGA and TAA became CGA and CAA respectively. These are examples of U to C type RNA editing, which was never been found before in chloroplast mRNA. In general, 13 Cs of genomic DNA were found as Ts in the cDNA sequence and seven Ts were found as Cs. This is the first finding of RNA editing on the transcripts of rbcL and also in bryophytes. This event had been thought to arise in land plants after the split of bryophytes. The origin of RNA editing is discussed in relation to the landing of green plants. - One serious challenge against the central dogma of molecular biology is the discovery of RNA editing. Genetic information not found in the genomic template can be transferred into mRNA after transcription. RNA editing was first discovered in the kinetoplast genetic system of trypanosome (1), later in the nuclear encoded mRNA of human apolipoprotein (2), and in a number of transcripts encoded by plant mitochondrial DNA (37). These events were found in all major groups of land plants except bryophytes (7). In angiosperm chloroplasts, RNA editing has also been identified (818). In chloroplasts, all RNA editing found so far has been C to U substitutions, whereas U to C substitutions have also been found in plant mitochondria. It had been thought * To whom correspondence should be addressed DDBJ accession nos D43695 and D43696 that editing arose in early land plants after the split of bryophytes because no editing has been identified in representatives of green algae and in liverwort (7). However, we found U to C RNA editing as well as C to U in rbcL transcripts of hornwort (Anthoceros formosae) chloroplasts. MATERIALS AND METHODS The following oligonucleotide primers designed from genomic DNA sequence of A. formosae were synthesized and obtained from Sawaday Technology (Tokyo, Japan) or Biologica (Nagoya, Japan): P1, 5-AGTAGACTTCGTCCCTGCAAGAGTT; P2, 5-TCCTCTCCAGCAACAGGTTCAATGT; P3, 5-AACTGGTACATGGACTACTGTTTGG; P4, 5-CTACTGTACCTGGATGAATATGATC; P5, 5-ACCGACAGACAAAGAAATCATGGTA; P6, 5-AAAACGAAAGAGCTGAATTGCAA; P7, 5-CCTCCTGTCAAATAATCATGCATTAC. Isolation of genomic DNA from chloroplast-rich fraction Thalli of the hornwort A.formosae were incubated at 25 C on 1/2 KnopII-agar medium under continuous fluorescent light. The thalli were harvested and homogenized in a buffer containing 50 mM TrisHCl (pH 8.0), 10 mM EDTA, 20% sucrose, 5 mM 2-mercaptoethanol, 0.1% BSA. The homogenate was filtered though cheese-cloth and unbroken cells were precipitated by centrifugation at 1000 g for 10 s. The chloroplast-rich fraction was precipitated from the supernatant by centrifugation at 3000 g for 10 min. Nucleic acids were extracted from the chloroplast-rich fraction as described by Dellaporta et al. (19). Contaminating RNA was removed from the DNA sample by digestion with RNaseA. Cloning of genomic DNA and identification of clones DNA from the chloroplast-rich fraction was partially digested with BglII and 1520 kb fragments were electro-eluted from an agarose gel as described (20). The fragments were then ligated to the Charomid 9-28 vector (Nippon Gene), packed in phage particles using Gigapack (Funakoshi), and infected into Escherichia coli DH5a . 100 colonies grown in the presence of ampicillin were transferred to Biodyne A membranes (Pall) and hybridized with a HindIII fragment containing part of the rbcL gene of Angiopteris lygodiifolia (21). The membranes were washed with 0.1 SSC containing 0.1% SDS at 50 C and exposed to Fuji X-ray film. Five positive clones, pCH13, pCH47, pCH48, pCH62 and pCH79, were identified. Plasmids were isolated by the boiling lysis (22) and analyzed by Southern hybridization using the HindIII fragment described above as the probe. A KpnI fragment of 7.5 kb was excised from pCH79 and ligated into pUC18. The resulting plasmid named pK79 was used for sequencing after subcloning into the pUC18 vector. Plasmids pCH13, pCH47, pCH48 and pCH62 were cut with SalI and religated. The five resulting plasmids were cut with HindIII, then 2.9 kb fragments were subcloned into pUC18 and sequenced by dideoxy chain-termination (23) using the BcaBest sequencing kit (Takara) or 7-deaza Sequenase Ver. 2.0 (USB). The products of the sequencing reactions were applied to a denaturing polyacrylamide gel and exposed to Fuji X-ray film. The resulting sequences were treated with Genetyx software Ver. 7.06 (SDC). Isolation of total RNA Total cellular nucleic acids were prepared using CTAB (24) with a slight modification. Frozen thalli (3 g) of A.formosae were disrupted with quartz sand and nucleic acids were extracted in 10 ml of extraction buffer containing 100 mM TrisHCl (pH 8.0), 20 mM EDTA, 1.4 M NaCl, 1% cetyltrimethylammonium bromide (CTAB) and 1% 2-mercaptoethanol at 60 C for 30 min. Total RNA was precipitated by adding LiCl to a final concentration of 2 M after extraction of chloroformisoamyl alcohol (24/1,v/v). Contaminating DNA was removed from the RNA sample by digestion with RNase-free DNase (Boehringer Mannheim). Reverse transcription of RNA The total RNA was used to synthesize cDNA using a commercial kit (lst-Strand cDNA Synthesis Kit, Clontech). Total RNA (0.5 m g) and 20 pmol random hexamer primer were annealed at 70 C for 2 min and cooled to 0 C. They were then mixed with 10 pmol of each of four nucleotide triphosphates (dNTP), 20 U recombinant RNase inhibitor and 200 U M-MLV reverse transcriptase in a total volume of 20 m l. The reaction proceeded at 42 C for 60 min and at 94 C for 5 min, then 80 m l of water was added. The cDNA was amplified by means of a PCR Amplification Kit (Takara) and the primer pairs P1P2, P3P4 and P5P6 (described above) by Program Temp Control System PC-700 (Astec). The reaction (100 m l) contained 10 m l of diluted cDNA, 100 pmol of each primer pair, 20 pmol of each of dNTP and 2.5 U Taq DNA polymerase. The thermocycles were 94 C for 2 min, 55 C for 2 min and 72 C for 2 min. Typically, the reaction proceeded though 30 cycles, followed by 72 C for 7 min. Amplified cDNAs were separated by agarose gel electrophoresis and extracted from the gel using Gene Clean II Kit (Bio 101). They were then filled-in with the Klenow fragment (Nippon Gene) and T4 polynucleotide kinase (Takara) (...truncated)