ALS pathophysiology–associated RBPs induce the production of a dominant-negative splicing isoform of TARDBP. (A) Schematic representation of alternative splicing isoforms of TARDBP and corresponding PCR product sizes. Black-framed arrows indicate the primer locations used for RT-PCR analysis of exons 5–7. For RT-qPCR, TaqMan MGB probes (magenta boxes) and primer sets specifically targeting TDP-FL or MP20 (127) transcripts are shown. (B) Representative RT-PCR results of TARDBP splicing in HEK293T cells overexpressing FLAG-RBPs, including ALS-causative RBPs (TDP-FL, FUS, and hnRNP A1), neuron-specific RBPs (ELAVL3 and NOVA1), and RBPs associated with ALS pathophysiology (hnRNP K, hnRNP E1, and hnRNP E2). A schematic of the sMP13 isoform is also provided in Fig. S1 F. (C) Quantification of MP20+ band ratios from B based on RT-PCR analysis. The band intensities were quantified from three independent experiments. (D) Relative proportions of MP20+ isoforms determined through sequencing analysis of PCR bands in cells with or without FLAG-hnRNP K overexpression. Analysis included 19 cloned colonies per condition. Dashed lines indicate the MP20+ proportion observed in the NoTF control. (E) RT-qPCR analysis of TDP-FL and MP20 (127) mRNA levels in HEK293T cells overexpressing FLAG-hnRNP K. Data are normalized to ACTB expression (n = 3 for each group). (F) Representative RT-PCR images of TARDBP splicing in HEK293T cells with hnRNP K KD. (G) RT-qPCR quantification of hnRNP K, TDP-FL, and MP20 (127) mRNA levels in hnRNP K KD cells. Data are normalized to ACTB expression (n = 3 for each group). Statistical analyses were performed using one-way ANOVA followed by Dunnett’s test (E) or Welch’s t test (G). All graphs display mean ± SEM. *P < 0.05 and ****P < 0.0001. (H) Schematic illustrating how hnRNP K promotes the splicing of TARDBP to generate the MP20 (127) isoform. Source data are available for this figure: SourceData F3.
Figure 3.

ALS pathophysiology–associated RBPs induce the production of a dominant-negative splicing isoform of TARDBP. (A) Schematic representation of alternative splicing isoforms of TARDBP and corresponding PCR product sizes. Black-framed arrows indicate the primer locations used for RT-PCR analysis of exons 5–7. For RT-qPCR, TaqMan MGB probes (magenta boxes) and primer sets specifically targeting TDP-FL or MP20 (127) transcripts are shown. (B) Representative RT-PCR results of TARDBP splicing in HEK293T cells overexpressing FLAG-RBPs, including ALS-causative RBPs (TDP-FL, FUS, and hnRNP A1), neuron-specific RBPs (ELAVL3 and NOVA1), and RBPs associated with ALS pathophysiology (hnRNP K, hnRNP E1, and hnRNP E2). A schematic of the sMP13 isoform is also provided in Fig. S1 F. (C) Quantification of MP20+ band ratios from B based on RT-PCR analysis. The band intensities were quantified from three independent experiments. (D) Relative proportions of MP20+ isoforms determined through sequencing analysis of PCR bands in cells with or without FLAG-hnRNP K overexpression. Analysis included 19 cloned colonies per condition. Dashed lines indicate the MP20+ proportion observed in the NoTF control. (E) RT-qPCR analysis of TDP-FL and MP20 (127) mRNA levels in HEK293T cells overexpressing FLAG-hnRNP K. Data are normalized to ACTB expression (n = 3 for each group). (F) Representative RT-PCR images of TARDBP splicing in HEK293T cells with hnRNP K KD. (G) RT-qPCR quantification of hnRNP K, TDP-FL, and MP20 (127) mRNA levels in hnRNP K KD cells. Data are normalized to ACTB expression (n = 3 for each group). Statistical analyses were performed using one-way ANOVA followed by Dunnett’s test (E) or Welch’s t test (G). All graphs display mean ± SEM. *P < 0.05 and ****P < 0.0001. (H) Schematic illustrating how hnRNP K promotes the splicing of TARDBP to generate the MP20 (127) isoform. Source data are available for this figure: SourceData F3.

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