Extended Data Fig. 8: The impact of variants at rs12973192, rs12608932 and rs56041637 on splicing.
From: TDP-43 represses cryptic exon inclusion in the FTD–ALS gene UNC13A
(a) Diagrams showing the design of the UNC13A minigene reporter constructs used to assess the impact of the variants at each locus. The complete design of the reporter construct is shown in Fig. 4d. For clarity, the mCherry and GFP exons that are closest to the promoter (blue in Fig. 4d) are labeled as exon 1, and the downstream exon are labeled as exon 2. REF is the reporter that carries the reference haplotype. M-1 to M-3 carry a single risk variant. M-4 to M-6 carry two risk variants. M-7 carries all three variants, the risk haplotype. (b, c) The locations of the RT-qPCR primer pairs used to detect the inclusion of the cryptic exon (b) and the splicing of EGFP (c, shown in black). (d, e) The expression level of the cryptic exon (d) or the splicing of EGFP (e) in each condition is calculated with reference to the expression level of cryptic exon or the splicing of EGFP from the WT construct in TDP-43-/- HEK-293T cells. The expression of the reporter construct measured using a pair of primers aligned to the second exon of EGFP (c, shown in green) was used to normalize RT-qPCR. The cryptic exon expression levels of each pair of reporters expressed within the same cell line were compared. The splicing of EGFP remained constant across all conditions, verifying equal reporter expression levels and the integrity of the splicing machinery independent of TDP-43.
