Fig. 5: Disrupting PTBP binding in SYNGAP1 intron 10 site upregulates SYNGAP1.
From: Mapping PTBP2 binding in human brain identifies SYNGAP1 as a target for therapeutic splice switching
a Western blot from HEK293T cells transfected for 48 h with siRNA against PTBP1 or PTBP2. siSC is a non-targeting control siRNA. b Top and left panel: RT-PCR from HEK293T cells transfected for 48 h with siRNA against PTBP1 or PTBP2 alone or in combination. Right panel: qPCR showing SYNGAP1 mRNA levels. c Top and left panel: RT-PCR from HEK293T cells transfected with 5 µM of PTBP decoy oligo for 48 h, including a non-targeting decoy (D_SCRM) as negative control. Right panel: qPCR showing SYNGAP1 mRNA levels. d Visualization of PTBP2 eCLIP-seq data from human cortex highlighting two highly enriched PTBP binding regions near SYNGAP1 exon 11: site 1 (green, intronic site) and site 2 (blue, NMD-inducing Exon 11x). Zoom-ins for both sites are provided including information about the nucleotide content (CU rich regions in red) and the location of the ASO walks. Light blue arrows indicate called peaks. e Scheme depicting 1-nt resolution ASO walk on SYNGAP1 site 1. The target region spans 33 nt of intronic sequence. Black lines denote introns, white rectangles denote constitutive exons and red rectangle denotes non-productive alternative exon (exon 11x). f RT-PCR from HEK293T cells transfected with 200 nM of ASO for 24 h, including a non-targeting ASO control (ET-SC) and no ASO control (Mock, −). g Top and left panel: RT-PCR from HEK293T cells transfected with increasing concentrations of lead ASO ET-019 and negative controls for 48 h. Right panel: qPCR showing SYNGAP1 mRNA levels. h Western blot from HEK293T cells transfected as in g. Data are represented as mean values ± SEM. All data points represent independent biological replicates. a, g, h (n = 3). b (n = 3 except n = 2 for siPTBP1 + siPTBP2). c (n = 4). f (n = 2). In a and b, one-way ANOVA with Dunnett’s multiple comparison test vs. siSC. c Student’s t test. g, h One-way ANOVA with Dunnett’s multiple comparison test vs. mock-treated cells (-). Source data are provided as a Source Data file. FC fold change.
