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Control of seed dormancy in Arabidopsis by a cis-acting noncoding antisense transcript
Monday, 2016/12/05 | 07:46:26
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Halina Fedak, Malgorzata Palusinska, Katarzyna Krzyczmonik, Lien Brzezniak, Ruslan Yatusevich, Zbigniew Pietras, Szymon Kaczanowski, and Szymon Swiezewski SignificanceSequential developmental transitions in plant life cycle are tightly controlled by dynamic regulation of key genes. Seed dormancy release is probably the first developmental transition in a plant’s life cycle, and it is regulated by the Delay of Germination 1 (DOG1) gene. Here we demonstrate that a nonprotein-coding antisense transcript originating from a conserved at DNA—but not protein level—DOG1 region is a negative regulator of DOG1 expression and seed dormancy establishment. We show that this antisense transcript negatively regulates DOG1 expression in cis. This mechanism is presumably conserved across the Brassicaceae, given the evolutionary conservation of the antisense DOG1 promoter. AbstractSeed dormancy is one of the most crucial process transitions in a plant’s life cycle. Its timing is tightly controlled by the expression level of the Delay of Germination 1 gene (DOG1). DOG1 is the major quantitative trait locus for seed dormancy in Arabidopsis and has been shown to control dormancy in many other plant species. This is reflected by the evolutionary conservation of the functional short alternatively polyadenylated form of the DOG1 mRNA. Notably, the 3′ region of DOG1, including the last exon that is not included in this transcript isoform, shows a high level of conservation at the DNA level, but the encoded polypeptide is poorly conserved. Here, we demonstrate that this region of DOG1 contains a promoter for the transcription of a noncoding antisense RNA, asDOG1, that is 5′ capped, polyadenylated, and relatively stable. This promoter is autonomous and asDOG1 has an expression profile that is different from known DOG1 transcripts. Using several approaches we show that asDOG1 strongly suppresses DOG1 expression during seed maturation in cis, but is unable to do so in trans. Therefore, the negative regulation of seed dormancy by asDOG1 in cis results in allele-specific suppression of DOG1 expression and promotes germination. Given the evolutionary conservation of the asDOG1 promoter, we propose that this cis-constrained noncoding RNA-mediated mechanism limiting the duration of seed dormancy functions across the Brassicaceae.
See: http://www.pnas.org/content/113/48/E7846.abstract PNAS November 29 2016; vol.113; no. 48: E7846–E7855
Figure: Fig. 1. The DOG1 3′ region is conserved at the DNA level (but the encoded polypeptide is not conserved) and colocalizes with an asDOG1 TSS. (A) DOG1 panel: schematic diagram of DOG1 gene organization. lgDOG1, long three exonic DOG1 transcript; shDOG1, short two exonic DOG1 transcript; STOP, end of ORF; black boxes, exon sequences; gray boxes, alternative exonic regions, sense and antisense transcripts are marked with arrows. DRS panel: reanalysis of polyA site mapping by Direct RNA sequencing. Reads mapped to the antisense strand represent sites where polyadenylation occurs (TTS antisense). VISTA panel: plot of a pairwise alignment between A. thaliana and A. lyrata DOG1 orthologs. The curve is calculated using default VISTA thresholds based on percentage identity shown on y axis and base pair position on the x axis (Materials and Methods). Regions longer than 100 bp with average conservation score above threshold of 70% were colored (exons in blue, introns and promoter in pink, and UTR in light blue). Alternative splicing is not marked. DNA alignment panel: multiple alignment of DOG1 orthologs from seven Brassicaceae members, colored according to percentage identity. Exon/intron annotation refers to the A. thaliana DOG1 gene. Stop codons are marked for each ortholog based on the in silico ORF prediction. (B) Motif analysis: DNA motifs in DOG1 orthologs, identified with MEME software. Only motifs with an e-value < 1 × 10−14 and P < 1 × 10−14 are shown. Exon annotation refers to the A. thaliana DOG1 gene. Alternative exon 2 is marked with a dotted line. Red boxes, motifs found in intron 2; blue boxes, motifs found in exon 3. (C) 5′ RACE sequencing results revealed two TSS of a 5′ capped antisense transcript originating from the end of exon 2. Individual DNA amplicons were cloned and sequenced. (D) RNA stability assay performed on Col-0 WT seedlings. Half-life (see table) was calculated based on degradation curves after cordycepin treatment (plot) for asDOG1, the sense shDOG1 transcript, the stable transcript of housekeeping gene EIF4A and a short-lived mRNA transcribed from gene At3g45970. Presented values are averages with SD from three independent experiments. |
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(44).png "Control of seed dormancy in Arabidopsis by a cis-acting noncoding antisense transcript")
