Fig 2: Replacement of the original CBP20 U12 intron by a U2 intron may lead to deleterious splicing events. (A) A table showing structures of three mini-gene constructs containing exons no. 4 and no. 5 from the A. thaliana CBP20 gene separated by the original U12 intron (construct no. 1), by the U2 intron from the Arabidopsis CBP80 gene (construct no. 6), and by the U2 intron from the P. sativum legumin gene (construct no. 7). (B) Qualitative RT-PCR analysis of splicing of mini-gene transcripts in tobacco protoplasts. Blue and green squares represent unspliced and spliced transcripts, respectively. The red square represents the alternatively spliced CBP80 U2 intron. (C) Quantitative analysis of mini-gene transcripts RT-PCR products using capillary electrophoresis. Splicing efficiency is calculated as a percentage of the sum of all detectable products. Values are shown as the mean ± SD (n = 3) from three independent experiments. (D) Nucleotide sequence of the mini-gene no. 6 containing the Arabidopsis CBP80 gene U2 intron. Green color depicts constitutive U2 5' and 3' splice site dinucleotides, red color represents an alternative 5' splice site, the STOP codon is marked in yellow, and blue color stands for the polypyrimidine tract. Asterisk – P < 0.005, Student’s t-tests.

Fig 3: The efficiency of CBP20 U12 intron splicing depends on intron length. (A) A table showing structures of five mini-gene constructs containing exons no. 4 and no. 5 from the A. thaliana CBP20 gene separated by one of the U12 introns differing in length and derived from different plant species. (B) Qualitative RT-PCR analysis of splicing of mini-gene transcripts in tobacco protoplasts. Blue and green squares represent unspliced and spliced transcripts, respectively. (C) Quantitative Real-time PCR analysis of mini-gene transcript splicing efficiency. Splicing efficiency of the mini-gene no. 1 transcript carrying the U12 intron from the Arabidopsis CBP20 gene was taken as 1. Blue bars represent fold change in splicing efficiency in comparison to the mini-gene no. 1 transcript. Red squares show the length of U12 introns derived from various plant species studied. Values are shown as the mean ± SD (n = 3) from three independent experiments. Asterisk – P < 0.001, Student’s t-tests.

Fig 4: Plant CBP20 U12 intron surrounded by four exons and two U2 introns is not efficiently recognized by the U12 splicing machinery. (A) A table showing structures of two midi-gene constructs containing A. thaliana or P. patens CBP20 gene fragment of the following composition: exon 3 – U2 intron 3 – exon 4 – U12 intron 4 – exon 5 – U2 intron 5 – exon 6. (B) Qualitative RT-PCR analysis of splicing of midi-gene transcripts in tobacco protoplasts. Arrows point to the improperly spliced products in which exons no. 4 and no. 5 are skipped. Green squares represent properly spliced midi-gene CBP20 transcripts. The rest of the bands above the main amplification products represent a variety of partially spliced transcripts. (C) Quantitative analysis of midi-gene transcript RT-PCR products using capillary electrophoresis. Splicing efficiency is calculated as a percentage of the sum of all detectable products. Values are shown as the mean ± SD (n = 3) from three independent experiments. Green color depicts properly spliced CBP20 mRNA isoform. (D) Structure of midi-genes and fully spliced transcript representing the most abundant splicing event.

Fig 5: CBP20 genes derived from different plant species present very similar gene structures and conserved localization of U12 introns. (A) Comparison of CBP20 gene structures. Green color depicts exons, yellow color marks U2 introns, red color indicates U12 introns, and orange color depicts the second exon of the CBP20 gene from S. moellendorffii, which is shorter than other exons in the same position in other plants studied by 3 nucleotides. Note that with the exception of S. moellendorffii, the exon lengths are the same in all plant species studied until exon no. 5 is reached, which is separated from the protein core-encoding part of the gene by the U12 intron. E – exon, i – intron. (B) A comparison of consensus elements of CBP20 U12 introns derived from the plant species studied (Simpson and Brown, 2008). Dashed lines represent individual nucleotide sequences between the 5' splice site and branch point. Dots mark alignment gaps. Nucleotides that differ from the consensus sequence in the branch point region are marked in black. The conserved adenine nucleotides in the branch point are marked in red.