Fmr1, Mtap1b, Sod1, Rhoa, and Calb1 mRNAs interact with FMRP in vivo. (A) Western blot analysis of UV-crosslinking and immunoprecipitation (CLIP) assay performed on Fmr1 wild-type (WT) and knock-out (KO) total brain lysates using polyclonal antibodies raised against the C terminus of FMRP. Input lysate (1/100th) and immunoprecipitates (IP, 1/20th) were probed for FMRP and its interacting protein partners FXR1P and FXR2P, respectively, by western blotting, revealing the presence of FMRP in the WT immunoprecipitates (upper panel revealed with 1C3 antibody), together with interacting partners FXR1P and FXR2P (lower panel revealed by 3Fx antibody). (B) RT-PCR analysis of mRNAs associated with FMRP. Total RNA was extracted from the input brain lysate and immunoprecipitates described in A, and used as a template for RT-PCR. RT-PCR products obtained from Fmr1-KO (lane 1) and WT (lane 2) inputs and from immunoprecipitates of Fmr1-KO (lane 3) and WT (lane 4) brain extracts were separated and visualized by agarose gel electrophoresis. This reveals that the known FMRP mRNA targets Fmr1 and Mtap1B are selectively recovered in the WT immunoprecipitate, while the unrelated mRNA Tubb3 is not recovered. Sod1 mRNA is also selectively recovered in the immunoprecipitate together with Rhoa and Calb1 mRNAs. Control PCR (lane 5) was performed in the absence of reverse-transcriptase. Lower DNA molecular weight markers presented on the left of the gels are, respectively, 100, 200, 300, 400, 500, 600, 800, and 1000 bp.
