Fig 1: RAVER1 and MBNL1 are required for full N1 exon repression, while DDX5/17 and TRA2B activate N1 exon splicing.(A) Diagram of the minigene splicing reporter. The WT N1 exon was inserted into the intron between constitutive exons 1 and 2 of human ß-globin (black boxes). The reporter was inserted into the Flp recombinase site of Flp-In HeLa cells (Life Technologies). Transcription of the reporter was induced by doxycycline. (B) SiRNA knockdown of splicing factors. The reporter cell line was transfected with the indicated siRNAs 48 hr before inducing reporter expression with doxycycline. Top panel: Total cellular RNA was harvested and assayed for reporter splicing by primer extension. Primer extension products were resolved on 4% urea-PAGE and autoradiographed. The exon-included and exon-skipped products are indicated, with the percent exon inclusion plotted below. Bottom panel: Averages of three experiments were plotted with standard deviations. p-values were calculated by comparing each knockdown to the control using a two-tail t-test. * = p<0.05; ** = p<0.01; **** = p<0.0001. (C) Immunoblots showing the efficiency of siRNA knockdown. Primer extension assays for siRNA set 2 is in Figure 5—figure supplement 1. GAPDH or ß-actin served as loading controls. The superscripted ‘F’ indicates fluorescent secondary antibodies used for detection.DOI: http://dx.doi.org/10.7554/eLife.19743.009