Figure 2.

Altered nad1 mRNA splicing in odb1. (A) Mitochondrial transcript splicing efficiencies were compared between the wild type (Col-0), odb1–1 and odb1–2 for all 23 mitochondrial introns, using a previously described qRT-PCR assay (16,22). This assay quantifies spliced transcripts by amplification of products that span splice junctions and unspliced transcripts by amplification of products that span exon-intron junctions. The histogram depicts splicing efficiencies as the ratio of spliced to unspliced products amplified from the mutant divided by the ratio of spliced to unspliced products amplified from the wild type, using a log₂ scale. Three technical replicates of two independent biological repeats were performed for mutant and wild-type plants for each transcript; standard errors are indicated. Transcript levels were additionally analysed for two intron-less mitochondrial genes (cox1, rps12); the nuclear 18S rRNA and ACT genes were used for data normalisation. (B) Probes for intron 1, intron 2, exon1, exons 2 and 3 (intron-spanning probe), and exon 5 of the mitochondrial nad1 gene were hybridised to filter-immobilised total RNA isolated from odb1–1, odb1–2 and wild-type (Col-0) seedlings (top panels). RNA marker sizes are indicated. Prior to hybridisations, membrane-bound RNAs were stained with methylene blue (bottom panels). Signals for the mature nad1 mRNA are indicated (m). Both odb1–1 and odb1–2 over-accumulate nad1 transcripts with introns 1 and 2 unspliced (u1,2). The exact composition of these transcripts is unclear as they were not detected with any of the exon-specific probes. The same holds true for a transcript of low abundance detected only in odb1–1 with the intron 2-specific probe (u2). The signal for this RNA was more pronounced in a similar experiment (Supplementary Figure S2). A transcript exclusively detected with probes for intron 1 and exons 2 and 3 and thus lacking exons 1 and 5 is reduced in odb1–1 (u1,3). This transcript also lacks exon 4 since no transcript of this size was detected with an exon 4-specific probe in an earlier study (58). (C) The nad1 gene has five exons that are joined by one cis- and three trans-splicing events. Lengths of exons and the cis-spliced intron are given in nucleotides; symbols are as in Figure 1. Dotted lines mark probe target regions.