Upstream SREs and a second GT at positions −8 and −7 affect the accuracy of TT splice site usage. (A) Overview of the used designer exon splicing reporter containing either five repeats of the splicing neutral sequence CCAAACAA (white boxes) or two SRSF7 binding sites (yellow box). The underlined bases represent CANC motifs, which arise from concatenating CCAAACAA and can serve as SRSF3 binding sites. (B) Sequences of the exon–intron border in the different designer exon variants. Lane numbers correspond to C and D. Potential SRSF3 binding sites are underlined. Sequence and HBond score of the U1 binding site at position −8 (bold GT) are shown below. Splicing registers at the noncanonical TT are indicated by −1 and +1. (C) HeLa cells were transfected with 1 µg of each of the depicted constructs and 1 µg of GH1, which was used as transfection control. Twenty-four hours post transfection, total RNA was isolated, reverse transcribed, and amplified with specific primer pairs: (DE) #2648/#2649; (S) splice site usage; (c1) c1 usage; (ES) exon skipping; (GH1) #1224/#1225. PCR products were separated either on a 10% PAA gel (bottom), or for higher resolution, on a QIAxcel DNA screening gel cartridge (top). (D) Sequencing results of the splice site usage shown in C. PAA bands were isolated, reamplified with the primer pair #2648/#2649, and sent to sequencing analysis using primer #2648. Blue shades in the sequencing chromatogram represent sequencing uniqueness, and black lines roughly indicate the level of alternative splice site usage. (E) The noncanonical CT splice site sequence CAG CTAAGTAT (cf. Figs. 1, 2) recruits SRSF3 in competition with U1 snRNA binding. In an RNA affinity chromatography assay, substrate RNAs containing a bacteriophage MS2 sequence and either canonical GT or noncanonical CT splice site sequences with otherwise full U1 snRNA complementarity were covalently linked to adipic acid dihydrazide-agarose beads (AB) and incubated with HeLa cell nuclear protein extract. Recombinant bacteriophage MS2 coat protein was added to monitor RNA input. Precipitated proteins were resolved by SDS-PAGE (15%) and detected by immunoblot analysis using anti-SRSF3, anti-SNRPC, or anti-MS2 coat protein antibodies.
