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 5Ј-and 3Ј-DNA flanking sequence has permitted the definition of specific signatures for each 5S rDNA array.

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.

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.

SummaryThe effect of alteration of 5¢ and 3¢¯anking sequences on the transcription of plant tRNA genes was analysed using an RNA polymerase III-dependent in vitro transcription system derived from nuclei of cultured tobacco cells. A TATA-like sequence and the CAA motif frequently observed upstream of plant tRNA genes, and the poly(T) stretch usually present downstream, were shown to be necessary for ef®cient re-initiation of transcription. The CAA motif was shown to be a transcription initiation site.

SummaryA nuclear extract derived from tobacco cultured BY-2 cells supports RNA polymerase Ill-dependent transcription of Ser ArabidopsistRNAgenes. Primer extension analysis indicated that the transcription starts at 6 bp upstream from the 5' end of tRNA coding region. Procedures for nuclear extraction and in vitro reaction conditions have been optimized for tRNA transcription, which allows direct detection of de novo synthesized tRNA by gel electrophoresis. This improved in vitro system yields a mature-sized tRNA of 85 n ucleotides from the Arabidopsis tRNA sar gene, indicating that efficient processing of the pre-tRNA also occurs in the tobacco nuclear extract.