hnRNP A1 inhibits the effect of hnRNP K on the alternative splicing of TARDBP. (A) Schematic representation of the mini-gene containing TARDBP exon 5 to exon 6 (1–2,100 nt) (mini-gene Ex5-Int5-Ex6) and the spliced PCR product length. Black-framed arrows indicate the primer locations for FLAG amplification to exon 7 of TARDBP. Exons and intron 5 (Int 5) are shown as white boxes with black lines, while the pale gray box with a dotted line represents the region that becomes exon 7 through alternative splicing. Orange and blue lines represent alternative donor (D) and acceptor (A) sites, respectively. (B) RT-PCR images showing TARDBP mini-gene splicing in cells overexpressing FLAG-hnRNP K and FLAG-hnRNP A1 at different doses. (C and D) RT-PCR images of endogenous TARDBP splicing (C) or RT-qPCR analysis of TDP-FL and MP20(127) mRNA levels (D) in HEK293T cells co-overexpressing FLAG-hnRNP K and FLAG-hnRNP A1. (E) WB analysis of HEK293T cells co-overexpressing FLAG-hnRNP K and FLAG-hnRNP A1. (F) Schematic diagram of hnRNP A1 RRM1 or RRM2 domain mutants (R1mt and R2mt). (G) RT-PCR images of endogenous TARDBP splicing in HEK293T cells coexpressing FLAG-hnRNP A1 mutants (R1mt or R2mt) with or without FLAG-hnRNP K. (H) RT-qPCR analysis of TDP-FL and MP20 (127) mRNA expression levels. In D and H, data are normalized to ACTB. Statistical analyses were performed using one-way ANOVA followed by Tukey’s test (n = 3 for each group). (I) WB analysis of HEK293T cells co-overexpressing FLAG-hnRNP A1 RRM domain mutants (R1mt or R2mt) and FLAG-hnRNP K. In E and I, quantification of relative expression levels of FL-endo, SVs-endo, and MP20-endo is shown (n = 3 for each group). Data are normalized to β-actin, and statistical analyses were performed using one-way ANOVA followed by Tukey’s test (E) or Dunnett’s test (I). All graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Gly-rich, glycine-rich region; RGG, arginine-glycine-glycine repetitive region. Source data are available for this figure: SourceData F7.
Figure 7.

hnRNP A1 inhibits the effect of hnRNP K on the alternative splicing of TARDBP. (A) Schematic representation of the mini-gene containing TARDBP exon 5 to exon 6 (1–2,100 nt) (mini-gene Ex5-Int5-Ex6) and the spliced PCR product length. Black-framed arrows indicate the primer locations for FLAG amplification to exon 7 of TARDBP. Exons and intron 5 (Int 5) are shown as white boxes with black lines, while the pale gray box with a dotted line represents the region that becomes exon 7 through alternative splicing. Orange and blue lines represent alternative donor (D) and acceptor (A) sites, respectively. (B) RT-PCR images showing TARDBP mini-gene splicing in cells overexpressing FLAG-hnRNP K and FLAG-hnRNP A1 at different doses. (C and D) RT-PCR images of endogenous TARDBP splicing (C) or RT-qPCR analysis of TDP-FL and MP20(127) mRNA levels (D) in HEK293T cells co-overexpressing FLAG-hnRNP K and FLAG-hnRNP A1. (E) WB analysis of HEK293T cells co-overexpressing FLAG-hnRNP K and FLAG-hnRNP A1. (F) Schematic diagram of hnRNP A1 RRM1 or RRM2 domain mutants (R1mt and R2mt). (G) RT-PCR images of endogenous TARDBP splicing in HEK293T cells coexpressing FLAG-hnRNP A1 mutants (R1mt or R2mt) with or without FLAG-hnRNP K. (H) RT-qPCR analysis of TDP-FL and MP20 (127) mRNA expression levels. In D and H, data are normalized to ACTB. Statistical analyses were performed using one-way ANOVA followed by Tukey’s test (n = 3 for each group). (I) WB analysis of HEK293T cells co-overexpressing FLAG-hnRNP A1 RRM domain mutants (R1mt or R2mt) and FLAG-hnRNP K. In E and I, quantification of relative expression levels of FL-endo, SVs-endo, and MP20-endo is shown (n = 3 for each group). Data are normalized to β-actin, and statistical analyses were performed using one-way ANOVA followed by Tukey’s test (E) or Dunnett’s test (I). All graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Gly-rich, glycine-rich region; RGG, arginine-glycine-glycine repetitive region. Source data are available for this figure: SourceData F7.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal