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

Figure 2. Effect of replacing the SLIII in U12 snRNA with the U6atac distal 3′ SL on in vivo splicing.

Figure 2

(a) Features of the branch site suppression assay. Wild type (WT) base pairing between human U12 snRNA and the branch site of the U12-dependent intron of P120 pre-mRNA is shown. The boxed nucleotides were mutated to their complementary nucleotides as shown. GA nucleotides at positions 23/24 in U12 snRNA were mutated to CU and the corresponding nucleotides UC at positions 84/85 in the branch site were mutated to AG. U12 GA23/24CU mutations are required to fully suppress the effect of the branch site UC84/85AG mutation. (b) Splicing phenotypes of P120 WT and the P120 UC84/85AG mutant coexpressed with the indicated U12 snRNA mutants. CHO cells were transfected with the indicated constructs and splicing phenotypes were assayed by RT–PCR. Lane M: 100 bp ladder. U12 GA23/24CU w U6atac SL denotes the U12 GA23/24CU snRNA construct containing the U6atac distal 3′ SL in place of U12 SLIII. 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 U12-dependent splice sites (U12 cryptic spliced) are indicated. The cryptic spliced product results from the activation of a U12-dependent cryptic splice site in the downstream exon. (c) Quantitative analysis of spliced/unspliced products. Numbers (x-axis) correspond to the respective lanes of the gel shown in (b). Error bars represent ± SE of three experiments.