Skip to main content
. 2021 Aug 24;220(10):e202101019. doi: 10.1083/jcb.202101019

Figure 6.

Figure 6.

Dependence of TDP-43 CR-mediated translation on physical interactions between TDP-43 and distinct translational factors. (A) Schematic overview of subcellular fraction and affinity purification strategies used in this study. After the removal of mitochondrial, nuclear, and unbroken cell pellets, the supernatant was subjected to the affinity purification of Strep-tagged TDP-43 by Strep-Tactin beads. The binding partners of TDP-43 were identified by LC-MS/MS. m/z, mass-to-charge ratio. (B) Volcano plots of TDP-43– and TDP-43ΔCR–interacting proteins detected by LC-MS/MS. The logarithmic ratios of average fold changes are shown on the x axis. The y axis data plot negative logarithmic false discovery rate (q) values from the t test performed on three biological replicates. Enriched proteins are denoted by red dots. (C) Venn diagram depicting the numbers of unique and shared interactors between TDP-43WT and TDP-43ΔCR. (D) Gene ontology (GO) enrichment analysis of unique and shared interactors in B according to categories based on biological processes. CRD,  coding region instability determinant; SRP, signal recognition particle. (E) Representative immunoblot of coIP between transfected Strep-Flag–tagged TDP-43WT/ΔCR and EGFP-PABPC4, Myc-RPS6, or Myc-RPL7 in HEK293 cells. Cells transfected with empty vector were also included as controls. Relative intensity quantifications are shown in the middle of the blots. (F) Representative immunoblot and quantification of coIP between transfected Strep-Flag–tagged TDP-43WT or ΔCR and endogenous PABPC4, RPS6, or PRL7 in HEK293 cells. (G) Representative immunoblot and quantification of coIP between endogenous TDP-43WT or ΔCR and endogenous PABPC4, RPS6, or PRL7 in the brains of WT and TDPΔCR+/− mice. IP using IgG was included as a control. (H) Schematic of TDP-43 with the highlighted amino acids from 80 to 107. (I) Representative immunoblot of coIP between transfected Strep-Flag–tagged TDP-43 (WT, ΔCR, or ΔCRΔ99-105) and endogenous PABPC4, RPS6, or PRL7 in HEK293 cells. (J) Representative immunoblot and quantification of newly synthesized proteins labeled by Click-iT AHA in TDP-43–knockout HEK293 cells expressing TDP-43WT, ΔCR, or ΔCRΔ99-105; n = 3 independent experiments per group). (K) Representative immunoblot and quantification of GFP expression driven by cap-dependent (CMV-EGFP) and cap-independent (IRES-EGFP) translation in TDP-43–knockout HEK293 cells expressing TDP-43WT, ΔCR, or ΔCRΔ99-105 (n = 3 independent experiments per group). (L) A working model of TDP-43 LLPS-regulated protein translation. WT TDP-43 forms droplet-like particles, while TDP-43ΔCR disrupts the phase transition process. Diffusive TDP-43ΔCR in cytoplasm enhances its interaction with translation factors involved in multiple translational steps, together causing translational abnormalities. Data are mean ± SEM; two-tailed Student’s t test (F and G), one-way ANOVA followed by Tukey’s multiple comparisons test (J). *, P < 0.05; **, P < 0.001; ***, P < 0.001; and ****, P < 0.0001.