Competition assay for determining noncanonical 5′ss efficiency. (A) Schematic of the HIV-1-based splicing reporter containing the different competitive splice site pairs. Sequence variations (denoted by “NN”) and HBond scores (https://www2.hhu.de/rna/html/hbond_score.php) of the competing canonical splice sites are indicated below. Enhancer repeats are highlighted in yellow (SRSF7) or blue (TIA1). (B,C) RT-PCR analyses of spliced reporter mRNAs. All splicing reporters in B contained SRSF7 splicing enhancer repeats, and the presence or absence of TIA1 repeats is indicated above each panel. Splicing reporters in C contained both SRSF7 and TIA1. (C) The comparison of two noncanonical 5′ss, inserted at the positions of “test” and “competing” 5′ss in A. (u) Upstream site; (d) downstream site. To monitor transfection efficiency, 2.5 × 105 HEK293T cells were transiently transfected with 1 µg of each: the respective HIV-1-based splicing reporter and pXGH5 (expressing human growth hormone 1 [GH1]). RNA was extracted 30 h post transfection and subjected to RT-PCR analysis as described in Methods. All experiments were performed in triplicates (Supplemental Fig. S6).
