Fig. 1: TDP-43 depletion in neurons leads to skipping of a constitutively expressed exon in the epilepsy gene, KCNQ2.
From: TDP-43-dependent mis-splicing of KCNQ2 triggers intrinsic neuronal hyperexcitability in ALS/FTD
a, Differential splicing analysis of stem cell-derived MNs treated with TDP-43 (n = 6) or scrambled (n = 6) siRNAs reported previously11. Annotated alternative splicing events are shown in green, de novo events are shown in orange and a de novo KCNQ2 event is shown in red. b, Mis-spliced genes after TDP-43 loss-of-function (LOF) involved in neuronal excitability. Left, integration of TDP-43 mis-spliced genes derived from purified MN11, i3Neurons9 and TDP-43-low neuronal nuclei41 yields 522 genes; 47 are involved in the regulation of ‘membrane potential’ or ‘synaptic signaling’. Right, two genes (KCNQ2 and CACNA1E) are mis-spliced in all three datasets. c, Sashimi plot depicting HISAT2-mapped sequencing coverage along exons 4–6 of KCNQ2. The gene model indicates the locations of exons and introns. Junction spanning reads mapping to the canonical junctions (exons 4–5 and 5–6) are displayed with solid lines, and de novo junction (exons 4–6) reads are displayed with a dashed line. Read counts and percent spliced index (PSI) scores for each splice junction are denoted. KCNQ2 exon skipping is only detected under TDP-43 depletion. siTDP-43, siRNA against TDP-43; siSCR, scrambled control siRNA. d, RT–PCR assay shows KCNQ2 mis-splicing in cortical neurons depleted of TDP-43 by siRNA. e,f, RT–qPCR of mis-spliced KCNQ2∆E5 that increases (e) and total KCNQ2 transcript that remains unaltered (f) after TDP-43 depletion. For e and f, circles represent n = 3 biological replicates, values represent mean expression; error bars, s.e.m. g–i, TDP-43 directly binds to KCNQ2 pre-mRNA. g, iCLIP sequencing coverage9 in the KCNQ2 locus suggests that TDP-43 binds KCNQ2. TDP-43 consensus motifs (UGNNUG) and (UG)n simple repeat are shown below the gene model. RIP amplicon used in h and i is indicated. h, Cross-linking immunoprecipitation followed by RT–PCR analysis shows enrichment of KCNQ2 in the TDP-43-bound fraction compared to IgG immunoprecipitates (IP) using the same quantity of lysate for IPs. i, Quantification of results present in h. Circles represent n = 3 biological replicates; values represent mean relative enrichment; error bars, s.e.m. For e, f and i, P values are the result of an unpaired two-sided t-test.
