Skip to main content
. 2015 Jun 5;43(13):6500–6510. doi: 10.1093/nar/gkv540

Figure 4.

Figure 4.

Analysis of Arabidopsis mitochondrial group II intron splicing pathways. The structures of released mitochondrial introns were analysed by RT-PCR performed on ligase-treated (lanes +L, cRT-PCR) and untreated (-L, RT-PCR) RNA extracted from Arabidopsis wild-type seedlings, using primers annealing to intron sequences as illustrated in Supplementary Figure S4. nad1 intron 2 (nad1i2; other nad gene introns are named accordingly) and nad2 intron 1 were additionally analysed for the odb1–1 mutant; results for these experiments are displayed in Supplementary Table S5. Gel images show products separated on 3% (w/v) agarose gels. Following cloning and sequencing of cRT-PCR and RT-PCR fragments, products labelled with black and grey arrowheads were identified to derive from introns released via lariat formation and first-step hydrolysis, respectively. Asterisks mark products that were found to be non-specific. dV lower stem models are shown below each gel image for the respective mitochondrial intron. To derive these models, homologous intron sequences from different plant species were aligned, putative dV regions were identified by folding conserved parts of the intron 3′ region (not displayed), and dVI stem-loops were then modelled from sequences downstream to dV. The mfold web server (59) was used for deriving putative dV and dVI secondary structures. Secondary structure models have previously been proposed for the four Arabidopsis nad5 introns, (60), nad7 intron 3 (61), nad1 intron 2, nad2 intron 1, nad2 intron 4 and nad4 intron 2 (34); they are identical with those derived here. dVI stems of introns that were found here to exclusively splice via the lariat pathway are depicted in black, and unpaired dVI nucleotides identified experimentally as lariat branch point are labelled with a filled black circle. dVI stem models of introns for which the use of a hydrolytic pathway was detected are depicted in grey. Grey stem models with unpaired nucleotides marked with a filled black circle correspond to introns for which the use of both a hydrolytic pathway and the lariat pathway was observed. Note that nad5 intron 4, which splices via the lariat pathway, failed to yield products from ligated RNA.