Figure 5.

DDX6 interacts with CUG-expanded DMPK-mRNA in vivo and in vitro. (A) RNA immunoprecipitation assay followed by semi-qRT-PCR analysis of FLAG-DDX6 immunoprecipitates reveals a strong binding preference of DDX6 for CUG-expanded DMPK-mRNA. Lanes 1–4 represents cDNA dilutions showing that PCR is conducted within the linear area of amplification. Lane 5 is DNA size marker. Lanes 6–9 show products of PCR reactions performed on the input cDNA before immunoprecipitation, which both demonstrates comparable loading and that DMPK-mRNA levels are virtually unaffected by either GFP (lane 6–7) or FLAG-DDX6 (lane 8–9) expression in both WT or DM1 fibroblasts. Lanes 10–13 shows the products from immunoprecipitated DMPK-mRNA for GFP negative control (lanes 10–11) and DDX6 (lanes 12–13). (B) Experiment was performed in triplicate and significance was determined by two-sided Student's t-test, where ‘***’ denotes P < 0.001). DDX6 IP-efficiency of GAPDH mRNA was included as a control and was quantified qRT-PCR, revealing that DDX6 precipitates GAPDH mRNA to similar levels in both WT and DM1 fibroblasts. (C) Western blots showing expression profile (INPUT—lanes 1 and 2) and IP-efficiency of FLAG-tagged DDX6 (lanes 3 and 4) used in the RIP experiment, demonstrating similar expression and IP-profiles between WT and DM1 cells. (D) Left panel: Band shift analysis using recombinant GST-DDX6 and CUG-200 RNA in binding reactions with increasing amounts of either DDX6 or DDX6(DEAA) mutant. Right panel: Competition experiment using high concentration of DDX6 or DDX6(DEAA) and cold CUG–RNA at 20-fold (+) or 500-fold (++) excess compared to radiolabeled CUG–RNA.