The accumulation of UNC13A cryptic RNA is sensitive to TDP-43 levels.

(A) Schematic representation of the UNC13A minigene constructs harboring the GWAS risk variants. The UNC13A minigene construct containing the human UNC13A cryptic exon sequence and the nucleotide flanking sequences upstream (50 bp at the of end of intron 19, the entire exon 20, the entire intron 20 sequence upstream of the cryptic exon) and downstream (remaining 857 bp intron 20 downstream of the cryptic exon) of the cryptic exon were expressed using the pTB vector. (B) Representative immunoblot (left) of cell lysates from each condition using an anti-TDP-43 C-terminal antibody and GAPDH as a loading control. Blots provided in Supporting information (S1 Raw images). Densitometric analysis (right) of TDP-43 protein levels, normalized to GAPDH, confirmed the reduction of TDP-43 in TARDBP KO HeLa cells compared to WT HeLa cells expressing either a control or TARDBP siRNA. (C) qRT-PCR showing TARDBP RNA levels in TDP-43 TARDBP KO compared to WT HeLa cells expressing a control siRNA or a siRNA against TARDBP. (D) qRT-PCR shown the enhancement of cryptic exon inclusion in UNC13A RNA (WT UNC13A minigene) in TARDBP KO cells, compared to WT cells treated with TARDBP siRNA. (E) qRT-PCR of TARDBP KO and WT HeLa cells expressing the different UNC13A minigene variants (shown in A) confirmed the accumulation of UNC13A cryptic RNA in TARDBP KO cells. Such accumulation was largest in cells containing the cryptic exon SNP (CE SNP and CE + intron SNP). All graphs represent mean ± SEM from 3 (B–D) or 2 (E) independent experiments. Statistical differences were assessed by one-way ANOVA followed by Tukeys’s (B–D) or Bonferroni’s (E) multiple comparisons test (ns: not significant, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001). Data used to generate graphs can be found in S3 Table. GWAS, genome-wide association study; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of mean; siRNA, small interfering RNA; SNP, single-nucleotide polymorphism; TDP-43, TAR DNA-binding protein-43.