TDP-43 RRM1 domain is necessary for including <i>POLDIP3</i> exon 3.

<p>(<b>A</b>) TDP-43 binds to <i>POLDIP3</i> mRNA. Whole cell lysate from HeLa cells was immunoprecipitated with anti-TDP-43 or control rabbit IgG, subjected to the isolation of RNA and RT-PCR analysis using primers located in exon 5 of <i>POLDIP3</i>. (<b>B–F</b>) Analysis of <i>POLDIP3</i> splicing by TDP-43 with mutated in RRM domains. (<b>B</b>) Schematic diagram of the constructs for wild-type (WT) and mutated TDP-43 expression plasmid (mRRM1, mRRM2, and mRRM1/2). Phe (F) to Leu (L) alterations were introduced in RRM1, RRM2, or both motifs of the TDP-43. Boxes indicate RRM motif. Numbers indicate the location of substituted amino acids. (<b>C</b>) TDP-43 depletion caused by siRNA targeting 3′-UTR of <i>TDP-43</i> (TDP-43 3′-UTR siRNA) was rescued by ectopic expression of WT and mutated TDP-43 in HEK293T cells. Mutated TDP-43s were similarly expressed. HEK293T cells were transfected with control (lane 1) or TDP-43 siRNA targeting for 3′-UTR region (lanes 2–6), followed by transfection with empty vector (EV) or various TDP-43 expression plasmids. TDP-43 expression was assessed by probing with anti-TDP-43 antibody. Anti-actin immunoblotting served as a loading control. (<b>D</b>–<b>F</b>) <i>POLDIP3</i> mRNA levels in culture cells transfected with <i>TDP-43</i> 3′-UTR siRNA as a fold change relative to those of cells transfected with control siRNA. The amounts of variant-1 (<b>D</b>), variant-2 (<b>E</b>) or total <i>POLDIP3</i> mRNA (<b>F</b>) were quantified by real-time qRT-PCR using the primers indicated with arrows in Fig. 2A. In this experiment, <i>RPLP1</i> and <i>RPS18</i> were used as reference genes. Data represent the mean with standard error from three independent experiments. Asterisk indicates significant difference among <i>TDP-43</i> 3′-UTR siRNA-transfected cells (*<i>P</i><0.01, Tukey multiple comparison test).