Takahiko Tsudzuki

Comparative analysis of RNA editing sites in higher plant chloroplasts.

Abstract. Transcripts of land plant chloroplast genomes undergo C-to-U RNA editing. Systematic search disclosed 31 editing sites in tobacco, 27 in maize, and 21 in rice. Based on these identified sites, potential editing sites have been predicted in the transcripts from four angiosperm chloroplast genomes which have been completely sequenced. Most RNA editing events occur in internal codons, which result in amino-acid substitutions. The initiation codon AUG was found to be created from ACG by RNA editing in the transcripts from rpl2, psbL, and ndhD genes. Comparison of editing patterns raises a possibility that many editing sites were acquired in the evolution of angiosperms.

The genomics of land plant chloroplasts: Gene content and alteration of genomic information by RNA editing

The entire nucleotide sequence of the chloroplast genome has been determined from 12 land plants. The gene content and arrangement are relatively uniform from species to species, and the genome contains an average of 111 identified gene species (except Epifagus). Chloroplast genes can be classified into three main categories: Genes for the photosynthetic apparatus, those for the transcription/translation system, and those related to biosyntheses. The genes encoding components of the photosynthesis apparatus have been identified by protein chemical analyses from higher plants, Chlamydomonas and cyanobacteria, and then by chloroplast transformation techniques using tobacco and Chlamydomonas. The genes for subunits of RNA polymerases and of ribosomes were initially deduced similarity to those in E. coli, and later confirmed by protein analyses. Coding information is often modified at the level of transcripts by RNA editing (mostly C-U changes), resulting in amino acid substitutions and creation of novel reading frames. Perspectives of chloroplast genomics are discussed.

RNA editing sites in tobacco chloroplast transcripts: editing as a possible regulator of chloroplast RNA polymerase activity

Abstract Genetic information in chloroplast DNA is sometimes altered at the transcript level by a process known as RNA editing. Sequence analysis of amplified cDNAs for 69 potential editing sites revealed 13 real editing sites in transcripts of 11 tobacco chloroplast genes. Together with those reported previously, these bring the total of edited sites observed in tobacco chloroplast transcripts to 31 (all involve C to U conversion). Alignment of sequences around the 31 editing sites revealed no obvious consensus, apart from an apparent bias for U or C at position −1 and A at position +2. Editing in tobacco rpoA mRNA restores the conserved leucine residue which is known to be important for transcriptional activation of the α subunit of E. coli RNA polymerase. Editing of this site is partial and the extent of editing depends on developmental conditions, suggesting that editing is, at least in part, involved in the regulation of chloroplast-encoded RNA polymerase activity.

Chloroplast DNA of black pine retains a residual inverted repeat lacking rRNA genes: nucleotide sequences of trnQ, trnK, psbA, trnI and trnH and the absence of rps16.

SummaryA physical map of black pine (Pinus thunbergii) chloroplast DNA (120 kb) was constructed and two separate portions of its nucleotide sequence were determined. One portion contains trnQ-UUG, ORF510, ORF83, trnK-UUU (ORF515 in the trnK intron), ORF22, psbA, trnI-CAU (on the opposing strand) and trnH-GUG, in that order. Sequence analysis of another portion revealed the presence of a 495 by inverted repeat containing trnI-CAU and the 3′ end of psbA but lacking rRNA genes. The position of trnI-CAU is unique because most chloroplast DNAs have no gene between psbA and trnH (trnI-CAU is usually located further downstream). Black pine chloroplast DNA lacks rps16, which has been found between trnQ and trnK in angiosperm chloroplast DNAs, but possesses ORF510 instead. This ORF is highly homologous to ORF513 found in the corresponding region of liverwort chloroplast DNA and ORF563 located downstream from trnT in Chlamydomonas moewusii chloroplast DNA. A possible pathway for the evolution of black pine chloroplast DNA is discussed.

Updated Gene Map of Tobacco Chloroplast DNA

The choroplast DNA from tobacco (Nicotiana tabacum) has often served as areference for plastid genomes. It is now analyzed extensively due to advancesin transplastome techniques and in vitro technology. The complete nucleotidesequence and gene map was published in 1986 (Shinozaki et al., 1986a,b).Since then, 24 new genes have been identified and sequencing errors havebeen found. Genes found after 1986 include: one small RNA gene (sprA)and 23 protein-coding genes(psaC, psaI, psaJ, psbI, psbJ, psbK, psbL, psbM,psbN, psbT, petG, petL, ndhG, ndhH, ndhI, ndhJ, ndhK, rp132, rp136, rpoC2,matK, accD and clpP). Figure 1 shows the updated gene map which includes105 different genes and 9 ycfs.Some of the sequence errors were found by our group (e.g. Shimada etal., 1990), and the remaining errors were reported and suggested by othergroups (e.g. Olmstead et al., 1993). We have examined these errors by re-sequencing. All corrections (Table 1) were made to the original sequenceand the numbering system was changed accordingly. The updated gene list(Table 2) and sequence have been deposited with the EMBL Nucleotide Se-quence Database under the accession number Z00044. A printed sequence inwhich genes/ycfs/orfs (see Figure 1) are boxed is available upon request toM. Sugiura.

Occurrence of silent RNA editing in chloroplasts: its species specificity and the influence of environmental and developmental conditions.

We have identified three new C-to-U RNA editing sites, one in atpF and two in atpA transcripts from tobacco chloroplasts. Two of them lead to amino acid substitutions to restore the conserved amino acid found in the corresponding genes of other plants. However, one editing site in the atpA transcript was found to take place partially at the third base of a serine codon (CUC to CUU), thus not leading to an amino acid substitution. This is the first report of silent editing in chloroplasts. The extent of silent editing depends on plastid stage and light conditions, while editing at another site (found 4 nt upstream from the silent editing site) takes place constitutively even in non-photosynthetic cultured cells and bleached white seedlings grown in the presence of spectinomycin and streptomycin. In pea and spinach, despite a conservation in sequence, no editing at the site corresponding to the silent site in tobacco was found. This observation suggests that the silent editing detected in this study is species-specific.

The 2005 version of the chloroplast DNA sequence from tobacco (Nicotiana tabacum)

The complete nucleotide sequence of tobacco chloroplast DNA was first determined in 1986, and then its updated gene map was reported in 1998. During the course of sequencing the chloroplast DNA ofNicotiana sylvestris, the female progenitor of tobacco, we found some sequence errors and amended the 1998 version. The tobacco chloroplast DNA comprises 155,943 bp, 4 bp longer than the 1998 version.

A novel hypoxic stress-responsive long non-coding RNA transcribed by RNA polymerase III in Arabidopsis

Recently, a large number of non-coding RNAs (ncRNAs) have been found in a wide variety of organisms, but their biological functions are poorly understood, except for several tiny RNAs. To identify novel ncRNAs with essential functions in flowering plants, we focused attention on RNA polymerase III (Pol III) and its transcriptional activity, because most Pol III-transcribed RNAs contribute to key processes relating to cell activities, and have highly conserved promoter elements: upstream sequence elements, a TATA-like sequence, and a poly(T) stretch as a transcription terminator. After in silico prediction from the Arabidopsis genome, 20 novel ncRNAs candidates were obtained. AtR8 RNA (approx. 260 nt) and AtR18 RNA (approx. 160 nt) were identified by efficient in vitro transcription by Pol III in tobacco nuclear extracts. AtR8 RNA was conserved among six additional taxa of Brassicaceae, and the secondary structure of the RNA was also conserved among the orthologs. Abundant accumulation of AtR8 RNA was observed in the plant roots and cytosol of cultured cells. The RNA was not processed into a smaller fragment and no short open reading frame was included. Remarkably, expression of the AtR8 RNA responded negatively to hypoxic stress, and this regulation evidently differed from that of U6 snRNA.

A new gene encoding tRNAPro (GGG) is present in the chloroplast genome of black pine: a compilation of 32 tRNA genes from black pine chloroplasts

The chloroplast genome of black pine (Pinus thumbergii), a gymnosperm, contains 32 different tRNA genes, 30 of which correspond to those previously identified in tobacco and rice chloroplast genomes. Two additional genes encode tRNAPro (GGG) and tRNAArg (CCG); the former is newly identified while the latter is present in liverwort, Physcomitrella patens and Angiopteris lygodiifolia, chloroplast genomes. Moreover, a partial copy of the split tRNAGly (UCC) gene and full copies of tRNAHis (GUG), tRNAThr (GGU) and tRNASer (GCU) genes are present in the large single-copy region of the genome, suggesting extensive rearrangements of the chloroplast genome during evolutio. No tRNA genes whose tRNA products can recognize codons CUU/C (Leu) and GCU/C (Ala) have been found. We propose that the 32 tRNAs are sufficient to read all the 61 sense codons in the black pine system using the “two-out-of-three” and the “U:N wobble” mechanisms.

Newly Identified Genes in Tobacco Plastid Genome

Our group first published the complete nucleotide sequence and gene map of tobacco chloroplast DNA in 1986 (1,2). After the publication, many studies on the sequence are performed to analyze the embedded information such as identification of new genes, determination of transcriptional units (3), surveillance of RNA editing sites (4) and revision of the sequence itself Our group found some sequence errors after the publication and we have examined errors reported and suggested by other groups (5) also by our re-sequencing. We summarize here the newly identified genes and updates in the sequence during past 12 years, and RNA editing sites so far determined.