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

Abstract

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.

Figure 1

Karyotype of Arabidopsis thaliana Columbia ecotype showing the loci of the 5S rDNA arrays in the pericentromeric regions of chromosomes 3, 4, and 5. The corresponding mapped CIC YACs and BACs for each 5S locus are presented.

Figure 2

Positions of the primers used for RT-PCR and PCR experiments. (A) Sequence of 5S rDNA-transcribed region whose size is 121-bp long and primers RT1-RT2. (B) Two consecutive 5S rDNA units are represented; PCR products are expected to be 495 bp with Oldg3-Oldg4 and Oldg5-Oldg6 and 491 bp with Oldg14-Oldg15.

Figure 2

Positions of the primers used for RT-PCR and PCR experiments. (A) Sequence of 5S rDNA-transcribed region whose size is 121-bp long and primers RT1-RT2. (B) Two consecutive 5S rDNA units are represented; PCR products are expected to be 495 bp with Oldg3-Oldg4 and Oldg5-Oldg6 and 491 bp with Oldg14-Oldg15.

Figure 3

Alignment of 5S RNA transcripts. Sequence of the major 5S RNA transcript is given in the top. Underlined residues of the major transcript correspond to differences found in the minor transcripts. Minor transcripts are indicated with the position and the substitute residue. Dots (.) indicate identical residues in the minor sequences. Further information is given on the right. The first column indicates tissues and/or developmental stages of the transcript cited. The second column indicates DNA correspondence of some transcripts (Chr4 = Chromosome 4 and Chr5 = Chromosome 5 major block). The third column indicates some 5S RNA found in polysomes.

Figure 4

Southern blot of yeast-recombinant DNA probed with 5S rDNA. Lanes 1 and 2 are EcoRI digestion of YAC 4E4 and 6A1, respectively. Lanes 3 and 4 are Bam H1 digestion of YACs 4E4 and 6A1, respectively.

Figure 5

Alignment of the transcribed region consensus sequences of 24 sequences from chromosome 5 minor block, 60 sequences from locus 2, 15 sequences from locus 1, and seven sequences from locus 3 of chromosome 3 with the major 5S RNA transcript. Dots (.) indicate identical residues. Different residues are underlined in the major 5S RNA sequence.

Figure 6

(A) Alignment of the transcribed region consensus sequences of chromosome 5 minor and major block, chromosome 4, and loci 1, 2, and 3 of chromosome 3. Transcribed region is between brackets. TATA-like sequence, GC sequence, C residue at −1, Box A, IE (Intermediate Element) and Box C are shaded.(B) Alignment of the 3′-flanking sequence of the transcribed region of chromosome 5 minor and major blocks, chromosome 4, loci 1, 2, and 3 of chromosome 3. Putative pol III termination site is shaded.

Figure 7

In vitro transcription.

Figure 8

Organization of 5S units in chromosome 5 major block. Each rectangle represents the 120-bp transcribed region. The chromosome 5 major block is fragmented in three domains: the YAC 6A1-restricted units, the central domain shared by YACs 6A1 and 4E4, and the YAC 4E4-restricted units. Only the most represented mutations have been indicated.