FXR1P selectively binds <i>in vitro</i> to the distal portion of <i>p21</i> mRNA 3′ UTR and associates <i>in vivo</i> with <i>p21</i> mRNA.

<p>(A) Scheme of the various portions of <i>p21</i> mRNA 3′ UTR (α, β and γ) used for <i>in vitro</i> binding assays. Note that the α fragment contains a characterized ARE motif. (B) Nitrocellulose filter binding assays to determine the portion of <i>p21</i> mRNA bound by FXR1P. Radiolabeled mRNA probes were incubated with increasing concentrations of recombinant FXR1P Isoe protein, the amount of radioactive probes recovered on filters after binding reaction is then plotted against the concentration of proteins. The portion of <i>FMR1</i> mRNA called N19 (known to be bound by FXR1P) and its truncated version (N19Δ35) were used as controls. This reveals that the distal portion of <i>p21</i> 3′UTR (γ fragment) and N19 are selectively bound by FXR1P. Both the α and β fragments from the 3′UTR of <i>p21</i> remain at background levels comparable to N19Δ35 binding to FXR1P. (C) Western-blot analysis of UV-crosslinking and immunoprecipitation (CLIP) assay performed on C2C12 lysates using polyclonal antibodies raised against the C-terminus of FXR1P (#830) and control rabbit IgG (R). Input lysates (lanes 1 & 2, Input, 1/50^th), immunoprecipitates (lanes 3 & 4, IP, 1/5^th) and post-immunoprecipitation supernatants (lanes 5 & 6, post, 1/50^th) were probed for FXR1P using the 3FX antibody. A selective enrichment in FXR1P medium and long isoforms is observed in #830 immunoprecipitate (lane 3), concomitant with a depletion in these isoforms in the post-immunoprecipitation supernatant (lane 4) as compared to corresponding controls (lane 3 & 5). (D) RT-PCR analysis of mRNAs associated with FXR1P complexes. RNA was extracted from input and immunoprecipitate fractions described in (C), and used as template for RT-PCR. RT-PCR products obtained from inputs and immunoprecipitations respectively from control with rabbit IgG (Lanes 1, 3) and immunoprecipitation of FXR1P using #830 (Lanes 2, 4) were separated and visualized by agarose gel electrophoresis. This reveals that <i>p21</i> mRNA is selectively enriched in the #830 immunoprecipitates, while the mRNAs encoding the myogenic determination factors Myogenin and MyoD or the unrelated mRNA encoding β-tubulin are not recovered in any immunoprecipitates. The symbol # indicates aspecific PCR products corresponding to <i>β-tub</i> primers dimers. DNA molecular weight markers presented on the gels are respectively 100, 200, 300, 400, 500, 600, 800 and 1000 bp.