The Tsta3 branchpoint sequence efficiently interacts with the U2 snRNA. (A) Representative gels of RT-PCR experiments analyzing the amount of U12 and U2 snRNAs binding to the beads alone (control beads), to the reporter RNAs without the UV cross-linking step or cross-linked to the Tsta3 (1) or Tsta3 BPS Slc12a4 (2) reporter pre-mRNAs. PCR reactions specific for sequences of the 3′ exon present in both reporters (E2) was used to normalize for affinity purification efficiency. The size (in base pairs) of the amplified fragments is indicated. (B) Relative enrichment of U12 and U2 snRNAs obtained with the control beads (CBs) and with the Tsta3 (1) or Tsta3 BPS Slc12a4 (2) reporter pre-mRNAs. For each snRNA, the intensities (expressed in arbitrary units) were normalized using the signal obtained with primers specific for the Tsta3 3′ exon (E2) as a reference. Statistical significance was calculated using the Student t-test (n = 4; (**) P < 0.01). (C) Semi-quantitative RT-PCR analyses of U2 snRNA amounts copurified with the Tsta3 (1) or Tsta3 BPS Slc12a4 (2) reporter pre-mRNAs after UV cross-linking. PCR reactions specific for sequences of the 3′ exon present in both reporters (E2) and used to normalize for affinity purification efficiency of the Tsta3 (1) or Tsta3 BPS Slc12a4 (2) reporter pre-mRNAs are shown. The size of the amplified fragments is the same as in A. The number of PCR cycles is indicated on the left. (D) Quantification of the PCR products shown in C. (E) Relative enrichment of U2 snRNA in UV cross-linking experiments performed with the Tsta3 (1) or Tsta3 BPS Slc12a4 (2) reporter pre-mRNAs. The intensities (expressed in arbitrary units) were normalized using the signal obtained with primers specific for the Tsta3 3′ exon (E2) as a reference (n = 2).
