Fig 2: TDP-43 binds directly to SKAR pre-mRNA. (A) Schematic representation of constructs used for RNA crosslinking assays. (B) SKAR DNAs comprising either exons 2–3 or exons 3–4 including intronic regions were in vitro transcribed/biotinylated and mixed with lysates from untransfected HEK293E cells or HEK293E cells transiently transfected with Flag-TDP-43 wt or ?RRM1/2. No RNA was added to control samples. Samples were UV crosslinked and precipitated with streptavidin-agarose. Western blots of streptavidin precipitates (left panel) were probed with anti-TDP-43 and anti-Flag to visualize co-precipitated endogenous and exogenous TDP-43. Biotinylated SKAR RNAs pulled down transfected as well as endogenous TDP-43 wt but not ?RRM1/2. Protein inputs (right panel) of HEK293E lysates confirmed equal transfection efficiencies. Note, that the used TDP-43 antibody does not recognize the TDP-43 ?RRM1/2 mutant as efficiently as TDP-43 wt because of partial deletion of the epitope. (C) Shorter SKAR fragments, as indicated in (A), were mixed to HEK293E lysates transfected with Flag TDP-43 wt. Anti-Flag and anti-TDP-43 probings of streptavidin pulldowns suggest part-5 to contain the preferential binding site within SKAR mRNA. Endogenous and transfected TDP-43 are indicated by closed and open arrowheads, respectively.

Fig 3: Validation of SKAR alternative splicing upon transient silencing of TDP-43. TDP-43 was either silenced transiently by siRNA treatment (A, C, E and G) or stably by use of lentiviral particles encoding for a TDP-43-specific shRNA followed by the selection of single cell clones (B, D and F). For transient silencing, HEK293E cells were either mock treated (m) or transiently transfected with scrambled control siRNA (scr), with one of four different TDP-43-specific siRNAs (siRNATDP-43 A-D) or with one of five specific siRNAs against FUS (siRNAFUS A-E), as indicated. (A–D) Total RNA was extracted and analyzed by RT–PCR. (A and B) Semi-quantitative RT–PCR was performed with primer pairs specific for TDP-43, SKAR (ex2–ex4), SKAR a (ex2|3–ex4) and SKAR ß (ex2|4–ex4). (C and D) Real-time PCR was performed with primer pairs against SKAR a (ex2|3–ex4) (white bars), SKAR ß (ex2|4–ex4) (gray bars) and total SKAR (ex5|6–ex7). PBGD was used as a housekeeping gene. Resulting relative SKARa/PBGD, SKARß/PBGD and total SKAR/PBGD ratios were recalculated into absolute copy values and normalized to total SKAR values. Shown are the mean values of five independent experiments ± SEM. *P < 0.05; **P < 0.005; ***P < 0.0005; ns = not significant. Original qRT–PCR data is presented in Supplementary Figure S1A and S1B, respectively. (E–G) Protein was extracted, electrophoresed and resulting western blots probed with antibodies specific for TDP-43, SKAR (both isoforms) and SKAR a. GAPDH was used as a loading control. FUS silencing efficiency was controlled by use of an anti-FUS antibody. Note, that, depending on the primer pair and antibody used, SKAR RNA and protein isoforms, respectively, are visualized as two bands with different molecular weights. The upper band represents SKAR a, the lower corresponds to SKAR ß, as indicated.

Fig 4: SKAR alternative splicing is dependent on RRM1 of TDP-43. (A) Stably silenced HEK293E cells (shRNATDP-43) or transiently silenced HEK293 cells (siRNATDP-43) were transiently transfected with either control vector (-) or Flag-TDP-43 variants (wt, ?RRM1, ?RRM2, ?RRM1/2, FFLL and ?GRD or disease-associated mutations, as indicated). Parental HEK293E cells or cells treated with a scrambled siRNA (-) were used as an internal control. (A) Total RNA was extracted and subjected to semi-quantitative RT–PCR using primer pairs amplifying total TDP-43, endogenous TDP-43, total SKAR (ex2–ex4), SKAR a (ex2|3–ex4), SKAR ß (ex2|4–ex4) and PBGD as a housekeeping gene. (B and E) RNA was extracted and real-time PCR performed with primer pairs against SKAR a (ex2|3–ex4) (white bars), SKAR ß (ex2|4–ex4) (gray bars) and total SKAR (ex5|6–ex7). PBGD was used as a housekeeping gene. Resulting relative SKAR a/PBGD, SKAR ß/PBGD and total SKAR/PBGD ratios were re-calculated into absolute copy values and normalized to total SKAR values. Original qRT data is presented in Supplementary Figure S1C and S1D, respectively. *P < 0.05; **P < 0.005; ***P < 0.0005; ns = not significant. (C and D) Protein was extracted, electrophoresed and resulting western blots probed with anti-TDP-43, anti-Flag and anti-SKAR antibodies. GAPDH was used as a loading control. (D) Shown are the mean values ± SEM of densitometric analysis of three independent experiments. *P < 0.05; **P < 0.005; ns = not significant.

Fig 5: GenomeGraph of SKAR as a splice target of TDP-43. HEK293E cells were transfected with control siRNA (scrambled) or treated with siRNA against TDP-43 (siRNATDP-43). Four biological replicates of each group were hybridized on a Human Exon 1.0-ST Gene Chip. Intensity values of microarray hybridizations, single values (gray), mean group intensities of scrambled siRNA (blue) and siRNATDP-43 (green), are shown as normalized background-corrected logarithmic intensities (A) and RMA corrected probe-level data (B). Vertical lines separate the 18 individual probe sets covering the POLDIP3/SKAR gene. (C) Depicted are the mean group values of the FIRMA score. The fold change of the FIRMA score (FC(F)) is shown in red. (D) Genomic representation of the POLDIP3/SKAR gene in orange. Gray lines at the top of this panel indicate localization of the individual probe sets within the genomic coordinates. (E) The two Ensembl annotated alternative splice isoforms SKAR a and SKAR ß are depicted in blue. SKAR exon 3 is highlighted by a box. (F) The SKAR a protein isoform is shown in pink, the RRM domain is shown in dark blue. Highlighted in green is the exon 3 derived part. At the bottom the amino acid sequence of exon 3 is given.