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Yasushi Yukawa

Publications and source records attributed to Yasushi Yukawa.

13 recordsLinked to original sources
PubMed2006

A survey of expressed tRNA genes in the chromosome I of Arabidopsis using an RNA polymerase III-dependent in vitro transcription system.

Eukaryotic tRNA genes are transcribed by RNA polymerase III. These tRNA genes are generally predicted using computer programs, and 620 tRNA genes in the Arabidopsis thaliana genome are currently annotated. However, no effort has been made to assay whether these predicted tRNA genes are all expressed, because it has been difficult to assay by routine in vivo methods. We report here a large-scale tRNA expression assay of predicted Arabidopsis tRNA genes using an RNA polymerase III-dependent in vitro transcription system developed by our group. DNA fragments including an annotated tRNA gene each were amplified by PCR and the resulting linear DNA was subjected to in vitro transcription. The addition of poly(dA-dT).poly(dA-dT) enhanced activity significantly and reduced background. The 124 predicted tRNA genes present in the Arabidopsis chromosome I were examined, and transcription activity and transcript stability from individual genes were determined. These results indicated that eight annotated genes are not expressed. Based on previous reports on pseudo-tRNA genes (e.g., Beier and Beier, Mol. Gen. Genet. 1992; 233: 201-208) and the present results, we estimated that 16% or more of the annotated tRNA genes in the chromosome I are not functional.

5' Flanking Region↗
PubMed2006

A simple in vitro RNA editing assay for chloroplast transcripts using fluorescent dideoxynucleotides: distinct types of sequence elements required for editing of ndh transcripts.

RNA editing is found in various transcripts from land plant chloroplasts. In tobacco chloroplasts, C-to-U conversion occurs at 36 specific sites including two sites identified in this work. Our RNA editing assay system using chloroplast extracts facilitated biochemical analyses of editing reactions but required mRNAs labeled with (32)P at specific sites. Here, we have improved the in vitro system using fluorescence-labeled chain terminators, ddGTP and ddATP, and have measured the editing activity at 19 sites in ndh transcripts. Editing activities varied from site to site. It has been reported that one editing site in ndhA mRNAs is present in spinach but absent in tobacco, but a corresponding editing capacity had been found in vivo in tobacco using biolistic transformation. We confirmed biochemically the existence of this activity in tobacco extracts. Using the non-radioactive assay, we examined sequences essential for editing within a 50-nt mRNA region encompassing an editing site. Editing of the ndhB-2 site requires a short sequence in front of the editing site, while that of the ndhF mRNA requires two separate regions, a sequence surrounding the editing site and a 5' distal sequence. These results suggest that distinct editing mechanisms are present in chloroplasts.

Chloroplasts↗
PubMed2006

Distinct modes of TATA box utilization by the RNA polymerase III transcription machineries from budding yeast and higher plants.

The TATA box is a key upstream control element for basal tRNA gene transcription by RNA polymerase III in some eukaryotes, such as the fission yeast (Schizosaccharomyces pombe) and higher plants, but not in others such as the budding yeast (Saccharomyces cerevisiae). To gain information on this differential TATA box requirement, we examined side-by-side the in vitro transcription properties of TATA-containing and TATA-mutated plant and S. cerevisiae tDNAs in homologous in vitro transcription systems from both organisms and in a hybrid system in which yeast TBP was replaced by its plant homologue. The data support the general conclusion that specific features of the plant transcription machinery, rather than upstream region architecture per se, are responsible for the much stronger TATA box dependence of the plant system. In both systems, however, a strong influence of the TATA box on transcription start site selection was observed. This was particularly striking in the case of plant tDNAs, where TATA-rich upstream regions were found to favour the use of alternative initiation sites. Replacement of yeast TBP with its plant counterpart did not confer any general TATA box responsiveness to the yeast transcription machinery. Interactions involving components other than TBP are thus responsible for the strong TATA box requirement of plant tDNA transcription.

Arabidopsis↗
PubMed2005

Plant 7SL RNA genes belong to type 4 of RNA polymerase III- dependent genes that are composed of mixed promoters.

The genes transcribed by RNA polymerase III (pol III) display a great diversity in terms of promoter structure and are placed in four groups accordingly. Type 3 subset of pol III genes has promoter elements which reside entirely upstream of the coding region of the gene whereas type 4 consists of genes with mixed promoters that enclose intra- and extragenic regulatory sequences. Plant 7SL RNA genes have been previously classified as type 3 of pol III genes requiring an upstream sequence element and a canonical TATA box for transcriptional activity in transfected plant protoplasts. We have identified two novel functional control regions within the coding region of an Arabidopsis 7SL RNA gene (At7SL-1) that resemble tRNA gene-specific A and B boxes with respect to sequence and position. Single and multiple nucleotide substitutions in either of these regions resulted in a pronounced reduction of transcription activity in tobacco nuclear extract that was not caused by a decreased stability as shown by decay kinetics of wild type and mutant RNA transcripts. These findings suggest that plant 7SL RNA genes should be actually placed in type 4 of pol III-transcribed genes. As a consequence of substantially different upstream promoters utilized by plant and human pol III, in vitro transcription of 7SL RNA genes in heterologous systems is severely impaired. A chimeric human 7SL RNA gene that contains the 5' flanking region up to position -300 of At7SL-1 is yet transcribed with a reduced efficiency in tobacco extract when compared with the plant wild-type gene, supporting the notion that internal regulatory elements contribute to full activity.

Arabidopsis↗
PubMed2003

Identification and characterization of transcription factor IIIA and ribosomal protein L5 from Arabidopsis thaliana.

Thus far, no transcription factor IIIA (TFIIIA) from higher plants has been cloned and characterized. We have cloned and characterized TFIIIA and ribosomal protein L5 from Arabidopsis thaliana. Primary sequence comparison revealed a high divergence of AtTFIIIA and a relatively high conservation of AtL5 when compared with other organisms. The AtTFIIIA cDNA encodes a protein with nine Cys(2)-His(2)-type zinc fingers, a 23 amino acid spacer between fingers 1 and 2, a 66 amino acid spacer between fingers 4 and 5, and a 50 amino acid non-finger C-terminal tail. Aside from the amino acids required for proper zinc finger folding, AtTFIIIA is highly divergent from other known TFIIIAs. AtTFIIIA can bind 5S rDNA, as well as 5S rRNA, and efficiently stimulates the transcription of an Arabidopsis 5S rRNA gene in vitro. AtL5 identity was confirmed by demonstrating that this protein binds to 5S rRNA but not to 5S rDNA. Protoplast transient expression assays with green fluorescent protein fusion proteins revealed that AtTFIIIA is absent from the cytoplasm and concentrated at several nuclear foci including the nucleolus. AtL5 protein accumulates in the nucleus, especially in the nucleolus, and is also present in the cytoplasm.

Amino Acid Sequence↗
PubMed2003

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.

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.

Cell Lineage↗
PubMed2003

A tRNA(Leu)-like sequence located immediately upstream of an Arabidopsis clock-regulated gene is transcriptionally active: efficient transcription by an RNA polymerase III-dependent in vitro transcription system.

A tRNA(Leu)-like sequence is located within a probable enhancer region of the RNA polymerase II-dependent gene encoding an RNA-binding protein, Atgrp7, in Arabidopsis (Mol. Gen. Genet. 261 (1999) 811). To examine whether this sequence is transcribed, we used our in vitro transcription system from tobacco cell nuclei. In vitro assays demonstrated that this tRNA-like sequence is transcribed by RNA polymerase III and its transcript is processed into tRNA-size molecules. Transcription starts at the CAA motif, a transcription initiation site for many plant tRNA genes. Mutation analyses indicated that transcription of this sequence depends on promoter elements typical for plant tRNA genes. We therefore concluded that this is a transcriptionally active tRNA(Leu)(AAG) gene. Mutation of a basic promoter element of the tRNA gene exerted no influence on the transcription of the downstream protein-coding gene, suggesting that no apparent interference occurs between the two adjacent genes.

5' Flanking Region↗
PubMed2003

In vitro analysis of the sequences required for transcription of the Arabidopsis thaliana 5S rRNA genes.

In vivo, we have already shown that only two of the 5S rDNA array blocks of the Arabidopsis thaliana genome produce the mature 5S rRNAs. Deletions and point mutations were introduced in an Arabidopsis 5S rDNA-transcribed region and its 5'- and 3'-flanks in order to analyse their effects on transcription activity. In vitro transcription revealed different transcription control regions. One control region essential for transcription initiation was identified in the 5'-flanking sequence. The major sequence determinants were a TATA-like motif (-28 to -23), a GC dinucleotide (-12 to -11), a 3-bp AT-rich region (-4 to -2) and a C residue at -1. They are important for both accurate transcription initiation and transcription efficiency. Transcription level was regulated by polymerase III (Pol III) re-initiation rate as in tRNA genes in which TATA-like motif is involved. Active 5S rDNA transcription additionally required an intragenic promoter composed of an A-box, an Intermediate Element (IE) and a C-box. Double-stranded oligonucleotides corresponding to different fragments of the transcribed region, used as competitors, revealed the main importance of internal promoter elements. A stretch of four T is sufficient for transcription termination. Transcription of Arabidopsis 5S rDNA requires 30 bp of 5'-flanking region, a promoter internal to the transcribed region, and a stretch of T for transcription termination.

3' Flanking Region↗
PubMed2002

Organization and transcription of the gene family encoding chlorophyll a/b-binding proteins in Nicotiana sylvestris.

Nine Lhcb1 genes encoding the light-harvesting chlorophyll a/b-binding proteins of photosystem II were isolated and characterized from Nicotiana sylvestris. Their nucleotide sequences are highly similar. Lhcb1 transcripts are accumulated in leaves and stems but not in roots and non-green cultured cells. RNase protection assay revealed that no transcripts were detected from the gene, Lhcb1*2, in Nicotiana tabacum. This finding raises the possibility that the amphidiploid tobacco cultivar (N. tabacum) lost one gene from the female progenitor (N. sylvestris) during evolution. Transcriptional initiation sites were mapped and found to be mostly cytidine residues, which is unique to the N. sylvestris Lhcb1 genes. Four of the nine genes have single start sites and the remaining genes possess multiple initiation sites. The TATA-like sequences of nine Lhcb1 genes can be classified into two groups; one that possesses a TTTATA sequence and the other that has a sequence diverged from it. The genes with single initiation sites belong to the first group. A consensus motif for the initiation region is CTC*A (C* for initiation site), which differs from those of other plant genes or mammalian genes.

5' Flanking Region↗
PubMed2002

Plant 7SL RNA and tRNA(Tyr) genes with inserted antisense sequences are efficiently expressed in an in vitro transcription system from Nicotiana tabacum cells.

RNA polymerase III-driven cassettes for the expression of antisense RNAs and ribozymes have recently attracted much attention because (1) pol III genes are transcribed abundantly in all kinds of tissues and (2) the transcripts are very stable by virtue of their small and compact size. We have designed two types of pol III-based expression vehicles. Antisense RNA sequences targeted against conserved structural elements or domains in the RNAs of potato spindle tuber viroid, hop latent viroid and potato virus S were either embedded in the anticodon region of a Nicotiana tRNA(Tyr) gene or near the 3' end of an Arabidopsis 7SL RNA gene. Both classes of chimeric genes were transcribed in vitro in a homologous plant extract. Our studies clearly revealed that the modified tRNA and 7SL RNA genes, carrying insertions of up to 90 and 120 bp, respectively, were expressed efficiently in the tobacco nuclear extract, resulting in high levels of stable chimeric transcripts. 7SL RNA (also termed SRP RNA) represents the RNA component of the signal recognition particle. This is the first report of demonstrating the employment of 7SL RNA genes as potential cassettes for the expression of antisense RNA and ribozyme sequences and might be helpful in future experiments to control their localization in specific sub-cellular compartments.

Base Sequence↗
PubMed2002

5S rRNA genes expression is not inhibited by DNA methylation in Arabidopsis.

Methylation has often been correlated with transcriptional inhibition of genes transcribed by polymerase II, but its role on polymerase III genes is less well understood. Using the genomic sequencing technique, we have analysed the methylation pattern of the different 5S-rDNA arrays of the Arabidopsis genome. Every cytosine position within the 5S sequence is highly methylated whatever the context - CpG, CpNpG or non-symmetrical. The methylation pattern of both transcribed and non-transcribed 5S units is similar, with no preferential methylated or unmethylated site. These results, taken together with 5-azacytidine treatments and in vitro transcription experiments using methylated 5S templates, demonstrate that 5S rRNA gene transcription is not inhibited by methylation. Non-transcribed 5S arrays are more subject to transition mutations resulting from deamination of 5-methylcytosines, leading to CpG depletions and an increasing A + T content. As there were no detectable differences in methylation, this implies more efficient repair and/or selection pressure in transcribed 5S-blocks.

Arabidopsis↗
PubMed2002

Analysis of the 5S RNA pool in Arabidopsis thaliana: RNAs are heterogeneous and only two of the genomic 5S loci produce mature 5S RNA.

One major 5S RNA, 120 bases long, was revealed by an analysis of mature 5S RNA from tissues, developmental stages, and polysomes in Arabidopsis thaliana. Minor 5S RNA were also found, varying from the major one by one or two base substitutions; 5S rDNA units from each 5S array of the Arabidopsis genome were isolated by PCR using CIC yeast artificial chromosomes (YACs) mapped on the different loci. By using a comparison of the 5S DNA and RNA sequences, we could show that both major and minor 5S transcripts come from only two of the genomic 5S loci: chromosome 4 and chromosome 5 major block. Other 5S loci are either not transcribed or produce rapidly degraded 5S transcripts. Analysis of the 5'- and 3'-DNA flanking sequence has permitted the definition of specific signatures for each 5S rDNA array.

Arabidopsis↗