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. 2016 Aug 11;6:31393. doi: 10.1038/srep31393

Figure 4. Combined effect of the exchange of SLs in both U6atac and U12 snRNAs on in vivo splicing.

Figure 4

(a) Features of the binary splice site suppression assay. In this assay, both branch site and 5′ SS mutations shown in Figs 2 and 3 were present in the P120 U12-dependent intron. The U12-dependent intron containing 5′ SS CC5/6GG and branch site UC84/85AG mutations is shown. The base pairing interactions between U11 GG6/7CC, U6atac GG14/15CC, U12 GA23/24CU snRNAs and the mutant 5′ SS and branch site of P120 intron are also shown. Boxed nucleotides denote the mutated nucleotides as shown in Figs 2 and 3. (b) Splicing phenotypes of P120 WT and the P120 CC5/6GG + UC84/85AG mutant coexpressed with the indicated suppressor snRNA constructs. CHO cells were transiently transfected with the indicated constructs and total RNA was extracted. The splicing pattern of the U12-dependent P120 intron was analyzed by RT-PCR using primers designed to bind flanking exons. Lane M: 100 bp ladder. The positions of bands corresponding to unspliced RNA, RNA spliced at the normal U12-dependent splice sites (U12 spliced) and RNA spliced at the cryptic U2-dependent splice sites (U2 cryptic) are indicated. (c) Quantitative analysis of spliced/unspliced products. Numbers on the x-axis correspond to the respective lanes of the gel shown in (b). Error bars represent ± SE of three experiments.