The branchpoint sequence is responsible for the Tsta3 splicing dependence on U2 snRNP. (A) Representative gel of RT-PCR experiments analyzing simultaneously the amount of unspliced and spliced products obtained with the Tsta3 reporter with different branchpoint sequences. Reporter names and size (in base pairs) of the amplified products are indicated on the left. The schematic structure of the splicing products is shown on the right. Preincubation of the splicing extracts with LNAs targeting the U2 (U2, 2 µM), U12 (U12, 2 nM), or U12 + U2 snRNAs is indicated. (B) Schematic representation and electrophoregram indicating the major cryptic splice sites activated in the Tsta3 BPS Slc12a4 reporter following partial inactivation of the U12 snRNPs. (C,D) Splicing efficiency (in %) determined for the Tsta3 BPS Slc12a4 (C) and Tsta3 BPS Mapk1 (D) reporter substrates following incubation in control (Ctl) or LNA-pretreated extracts. In C, Min refers to the regular splicing event and Maj to the cryptic one. (E) Representative gel of RT-PCR experiments analyzing simultaneously the amount of unspliced and spliced products obtained with the Tsta3 BPS Mapk1 reporter. Preincubation of the splicing extracts with LNAs targeting the U12 (U12, 2 µM) snRNAs is indicated. (F) Splicing efficiency (in %) determined for the Tsta3 BPS Mapk1 following incubation in control (Ctl) or LNA-pretreated extracts. Statistical significance was calculated using the Student t-test (n = 3; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001).
