Fig 2: 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 3: 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 4: SKAR ß is more active than SKAR a and leads to enhanced translation and increased cell size. (A) HEK293E cells were treated with control siRNA or transfected with siRNA against SKAR or TDP-43 as indicated. Stably silenced siRNATDP-43 and transiently transfected HEK293E cells were transfected with either control vector (-) or plasmids encoding for Myc-SKAR a, Myc-SKAR ß or Flag-TDP-43 wt, as indicated. Cells were serum starved for 16 h. After 6 h serum-stimulation cells were harvested, protein extracted and electrophoresed. Resulting western blots were probed with anti-SKAR, anti-phospho S6K1 (Thr389), anti-S6K1, anti-phospho S6 (Ser235/236), anti-S6, anti-phospho Akt substrate (RXRXXS/T) and anti-TDP-43 antibodies. GAPDH was used as a loading control. Transfection of SKAR ß or depletion of TDP-43 results in overall stronger phospho-signal compared to SKAR a. (B) Schematic representation of luciferase constructs used for analysis of translation. (C–G) HEK293E cells were transfected with either Myc-SKAR a or Myc-SKAR ß (C) or with control siRNA (scr) and individual siRNATDP-43 A–D, as indicated (D–G). (C–E) Before DNA/siRNA transfection, cells were transfected with firefly control vector plus either intron-containing or intron-less Renilla luciferase constructs. (C and D) Luciferase activity was measured and normalized to control treated HEK293E cells. Shown are the mean values ± SEM of five independent experiments. *P < 0.05. Western blotting confirmed equal expression of Myc-SKAR a and Myc-SKAR ß (C, right panel). (E) qRT–PCR confirmed equal RNA levels of Renilla and firefly luciferase in non-silenced and silenced HEK293E cells. (F) Cells were counted and equal numbers of cells was collected. Protein amount was determined using BCA protein assay. Shown are the mean values ± SEM of five independent experiments. *P < 0.05. (G) Cell size was analyzed by flow cytometry, monitoring the forward scatter parameter. Shown are the mean values ± SEM of five independent experiments. *P < 0.05; **P < 0.005.

Fig 5: A repeat containing RNA stretch 3' of exon 3 is necessary for TDP-43 and SKAR splicing. (A) Schematic representation of constructs used for refined RNA crosslinking assays. (B) Indicated fragments of SKAR DNA were in vitro transcribed/biotinylated and mixed with lysates form HEK293E cells transiently transfected with Flag-TDP-43 wt or FFLL. 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 FFLL. Protein inputs (right panel) of HEK293E lysates confirmed even transfection efficiencies. (C) Schematic representation of the three repeat motifs and mutagenized variants within the SKAR pre-RNA 3' of exon 3. (D) Non-mutated or mutagenized variants of SKAR DNA part-5 were in vitro transcribed/biotinylated and mixed with lysates form HEK293E cells transiently transfected with Flag-TDP-43 wt. No RNA was added to control samples. Samples were UV-crosslinked and precipitated with streptavidin-agarose. Western blots of streptavidin precipitates were probed with anti-TDP-43 and anti-Flag to visualize coprecipitated endogenous and exogenous TDP-43. (E) Schematic representation of the used SKAR minigene construct pTB SKAR part-3/4/5. Primer annealing sites are indicated by arrows. (F and G) HEK293E cells were transfected with pTB SKAR part-3/4/5 variants, as indicated. RNA was extracted and used for RT–PCR using primers for pTB and PBGD as a housekeeping gene. (F) Representative RT–PCR is shown. (G) Shown are the results (mean values ± SEM) of densitometric analysis of seven independent experiments calculated as the ratio of SKAR a to SKAR ß. *P < 0.05; ***P < 0.0005.