Fig. 5: TDP-43 directly binds to Btn1a1 and Xdh mRNA.
From: TDP-43 facilitates milk lipid secretion by post-transcriptional regulation of Btn1a1 and Xdh
a mRNA structures of Btn1a1 (upper) and Xdh (lower) showing TDP-43-binding motif in 3′-UTRs. Deep green boxes represent coding region. Light green boxes represent non-coding region. b, c RNA immunoprecipitation (RIP) assay for analysis of interaction between TDP-43 protein and Btn1a1 or Xdh mRNA in MECs and differentiated HC11 cell line using TDP-43 antibody. d, e MECs (d) and differentiated HC11 (e) cellular extracts were incubated with in vitro-transcribed biotin-labeled control (pcDNA3.1 vector), Btn1a1, or Xdh mRNA fragments for biotin RNA pull-down followed by western blot analysis. Control RNA used biotinylated RNA without TDP-43-binding site (ugugug). f Structures of TDP-43 and their mutants used in RIP assays. TDP-43 protein contains an N-terminal domain (N-term), two RNA-recognition motifs (RRM1 and RRM2), a glycine-rich domain (GRD), and a C-terminal domain (C-term). g Western blot of overexpressed Flag-TDP-43 or its mutants in HC11 cell line. h RIP assay using Flag antibody in differentiated HC11 cells after overexpression of Flag-TDP-43 or Flag-mutants of TDP-43. i Schematic of mouse Btn1a1 or Xdh mRNA fragments used for RNA pull-down assays. j Binding between TDP-43 protein and fragmented Btn1a1 and Xdh mRNA. Four biotinylated Btn1a1 mRNA fragments or six biotinylated Xdh mRNA fragments were incubated with HC11 cellular extract, and interaction between TDP-43 and each fragment was examined by RNA pull-down followed by western blot analysis. Data are shown as the means ± SD of three independent experiments. Source data are provided as a Source Data file.
