Yasushi Yukawa

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.

The complete structure of the cucumber (Cucumis sativus L.) chloroplast genome: Its composition and comparative analysis

The complete nucleotide sequence of the cucumber (C. sativus L. var. Borszczagowski) chloroplast genome has been determined. The genome is composed of 155,293 bp containing a pair of inverted repeats of 25,191 bp, which are separated by two single-copy regions, a small 18,222-bp one and a large 86,688-bp one. The chloroplast genome of cucumber contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species), and 37 tRNA genes (30 tRNA species), with 18 of them located in the inverted repeat region. Of these genes, 16 contain one intron, and two genes and one ycf contain 2 introns. Twenty-one small inversions that form stem-loop structures, ranging from 18 to 49 bp, have been identified. Eight of them show similarity to those of other species, while eight seem to be cucumber specific. Detailed comparisons of ycf2 and ycf15, and the overall structure to other chloroplast genomes were performed.

The downstream atpE cistron is efficiently translated via its own cis-element in partially overlapping atpB–atpE dicistronic mRNAs in chloroplasts

The chloroplast atpB and atpE genes encode subunits β and ε of the ATP synthase, respectively. They are co-transcribed as dicistronic mRNAs in flowering plants. An unusual feature is an overlap (AUGA) of the atpB stop codon (UGA) with the atpE start codon (AUG). Hence, atpE translation has been believed to depend on atpB translation (i.e. translational coupling). Using an in vitro translation system from tobacco chloroplasts, we showed that both atpB and atpE cistrons are translated from the tobacco dicistronic mRNA, and that the efficiency of atpB translation is higher than that of atpE translation. When the atpB 5′-UTR was replaced with lower efficiency 5′-UTRs, atpE translation was higher than atpB translation. Removal of the entire atpB 5′-UTR arrested atpB translation but atpE translation still proceeded. Introduction of a premature stop codon in the atpB cistron did not abolish atpE translation. These results indicate that atpE translation is independent of atpB translation. Mutation analysis showed that the atpE cistron possesses its own cis-element(s) for translation, located ~25 nt upstream from the start codon.

ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro.

Chloroplasts possess common biosynthetic pathways for generating guanosine 3′,5′-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5′-(β,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.

Translation of partially overlapping psbD-psbC mRNAs in chloroplasts: the role of 5′-processing and translational coupling

The chloroplast psbD and psbC genes encode the D2 and CP43 proteins of the photosystem II complex, and they are generally cotranscribed. We report studies on the basic translation process of tobacco psbD-psbC mRNAs using an in vitro translation system from tobacco chloroplasts. The primary transcript has an unusually long 5′-UTR (905 nt). We show that it is translatable. Processing of the 5′-UTR greatly enhances the translation efficiency of the psbD cistron. A striking feature is that psbD and psbC cistrons overlap by 14 nt. Removal of the psbD 5′-UTR plus the start codon and introduction of a premature termination codon in the psbD cistron considerably reduce the translation efficiency of the downstream psbC cistron. These results indicate that translation of the psbC cistron depends largely on that of the upstream psbD cistron and thus shows translational coupling; however, a portion is independently translated. These observations, together with the presence of monocistronic psbC mRNAs, suggest that the psbD and psbC cistrons are translated via multiple processes to produce necessary amounts of D2 and CP43 proteins.

Splicing of Arabidopsis tRNAMet precursors in tobacco cell and wheat germ extracts

Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNATyrGΨ A and elongator tRNAMetCmAU contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNATyr. Here we have studied the expression of an Arabidopsis elongator tRNAMet gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNAMet precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNAMet to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3′ and 5′ splice sites and of a structured intron for pre-tRNAMet splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNAMet splicing and that a highly structured intron is indispensable for pre-tRNAMet splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNAMet gene, is efficiently processed and spliced in both plant extracts.

Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA.

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.

A tRNALeu-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.

In Vitro Transcription Systems from BY-2 Cells

The in vitro transcription system is a powerful method to dissect molecular mechanisms of transcription of genes in question, usually performed in cellular or nuclear extracts containing transcription apparatuses with exogenous DNA templates and four nucleoside triphosphate (NTP) substrates. The first eukaryotic system was reported by Weil et al. (1979). They carried out in vitro transcription using a cytosolic extract and purified Pol II from HeLa cells. Manley et al. (1980) followed with in vitro assays using a whole cell extract from HeLa cells. In 1983, Dignam et al. (1983) established a standard procedure to prepare nuclear extracts from HeLa cells. Based on these efforts, many other in vitro systems have been established from different eukaryotic species, for example, yeast, mouse, and fruit fly. Since then, many significant discoveries have been made with these in vitro transcription systems.