The Plcb3 minor intron contains competing BPSs differentially affected by U2 depletion. (A) Schematic representation of the Plcb3 splicing reporter and electrophoregram indicating the alternative 3′ splice site used to generate the 127 nt mRNA. The U12 and U2 scores of the competing branch sites and the size of the exons and introns are indicated. Exonic sequences (gray boxes), minor splicing signals (splice donor, branchpoint sequence, and splice acceptor), as well as downstream sequences containing the major splice donor site are in capital letters. (B) Representative gel of RT-PCR experiments analyzing simultaneously the amount of unspliced and spliced products obtained with the Plcb3 reporter. Preincubation of the splicing extracts with LNAs targeting the U2 (U2, 2 µM), U12 (U12, 2 nM), or U12 + U2 snRNAs is indicated. The schematic structure of the splicing products is shown on the right and the size (in base pairs) is indicated on the left. (C) Splicing efficiency (in %) determined for the different splicing products following incubation in control (Ctl) or LNA-pretreated extracts. “Alt” refers to the 127-nt-long mRNA. Statistical significance was calculated using the Student t-test (n = 3; ns: nonsignificant; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001).
