ALS-associated mutant FUS exhibits a restricted capacity to suppress the expression of hnRNP K and MP20. (A) Comparison of sequences downstream of the FL-stop in exon 6 529–585 in TARDBP between humans and mice. Annotated with FL-stop (magenta), GU-rich (light blue), and AU-rich region (purple), respectively. Magenta lines and square box indicate the FUS-binding consensus reported previously. (B) Schematic representation of the FUS with P525L mutation. (C) RT-qPCR analysis of relative mRNA expression of MP20 (127), TDP-FL, and hnRNP K in HEK293T cells overexpressing FLAG-FUS. Data are normalized to ACTB. Statistical analyses were performed using one-way ANOVA followed by Tukey’s test (n = 3 for each group). (D) WB of HEK293T cells overexpressing FLAG-FUS. Quantification of relative expression of hnRNP K (n = 3 for each group). (E) Immunocytochemistry of HeLaS3 cells overexpressing FLAG-FUS. HnRNP K (green), FLAG (magenta), and Hoechst (blue). Arrowheads or arrows indicate FLAG-positive or FLAG-negative cells, respectively. Scale bar: 10 μm. Quantification of the fluorescence intensity of nuclear hnRNP K in FLAG-FUS–positive cells (right). (F) WB of HEK293T cells overexpressing FLAG-FUS. Quantification of relative expression of MP20-endo (n = 3 for each group). In D and F, data are normalized to β-actin. Statistical analyses were performed using one-way ANOVA followed by Tukey’s test. (G) Immunocytochemistry of HeLaS3 cells overexpressing FLAG-FUS. MP20 (green), FLAG (magenta), and Hoechst (blue). Arrowheads or arrows indicate FLAG-positive or FLAG-negative cells, respectively. Scale bar: 10 μm. Quantification of the fluorescence intensity of nuclear MP20 in FLAG-FUS–positive cells (right). In E and G, a total of 100 FLAG-positive and 100 FLAG-negative cells per group were analyzed. Fluorescence signals were normalized to the average intensity of 200 FLAG-negative cells. Statistical analysis was performed using Welch’s t test. (H) Comparison of sequences downstream of the MP20 stop codon in exon 6 1,198–1,246 in TARDBP between humans and mice. Annotated with MP20 stop codon (magenta) and GU-rich (light blue), respectively. The magenta lines and square box indicate the FUS-binding consensus reported previously. All graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Ab, antibody; RMM, RNA recognition motif; ZnF, zinc finger; NES, nuclear export signal; NLS, nuclear localization signal; QSYG, glutamine-glycine-serine-tyrosine-rich domain. Source data are available for this figure: SourceData F9.
Figure 9.

ALS-associated mutant FUS exhibits a restricted capacity to suppress the expression of hnRNP K and MP20. (A) Comparison of sequences downstream of the FL-stop in exon 6 529–585 in TARDBP between humans and mice. Annotated with FL-stop (magenta), GU-rich (light blue), and AU-rich region (purple), respectively. Magenta lines and square box indicate the FUS-binding consensus reported previously. (B) Schematic representation of the FUS with P525L mutation. (C) RT-qPCR analysis of relative mRNA expression of MP20 (127), TDP-FL, and hnRNP K in HEK293T cells overexpressing FLAG-FUS. Data are normalized to ACTB. Statistical analyses were performed using one-way ANOVA followed by Tukey’s test (n = 3 for each group). (D) WB of HEK293T cells overexpressing FLAG-FUS. Quantification of relative expression of hnRNP K (n = 3 for each group). (E) Immunocytochemistry of HeLaS3 cells overexpressing FLAG-FUS. HnRNP K (green), FLAG (magenta), and Hoechst (blue). Arrowheads or arrows indicate FLAG-positive or FLAG-negative cells, respectively. Scale bar: 10 μm. Quantification of the fluorescence intensity of nuclear hnRNP K in FLAG-FUS–positive cells (right). (F) WB of HEK293T cells overexpressing FLAG-FUS. Quantification of relative expression of MP20-endo (n = 3 for each group). In D and F, data are normalized to β-actin. Statistical analyses were performed using one-way ANOVA followed by Tukey’s test. (G) Immunocytochemistry of HeLaS3 cells overexpressing FLAG-FUS. MP20 (green), FLAG (magenta), and Hoechst (blue). Arrowheads or arrows indicate FLAG-positive or FLAG-negative cells, respectively. Scale bar: 10 μm. Quantification of the fluorescence intensity of nuclear MP20 in FLAG-FUS–positive cells (right). In E and G, a total of 100 FLAG-positive and 100 FLAG-negative cells per group were analyzed. Fluorescence signals were normalized to the average intensity of 200 FLAG-negative cells. Statistical analysis was performed using Welch’s t test. (H) Comparison of sequences downstream of the MP20 stop codon in exon 6 1,198–1,246 in TARDBP between humans and mice. Annotated with MP20 stop codon (magenta) and GU-rich (light blue), respectively. The magenta lines and square box indicate the FUS-binding consensus reported previously. All graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Ab, antibody; RMM, RNA recognition motif; ZnF, zinc finger; NES, nuclear export signal; NLS, nuclear localization signal; QSYG, glutamine-glycine-serine-tyrosine-rich domain. Source data are available for this figure: SourceData F9.

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