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.