Figure 4: HuR binds to the NPM mRNA via two U-rich sequences within the 3′-UTR.

(a) IP experiments were performed using the monoclonal HuR antibody (3A2), or IgG as a control, on total cell lysates from C2C12 cells. RNA was isolated from the immunoprecipitate, and RT-PCR or RT-qPCR was performed using primers specific for NPM and RPL32 mRNAs. The agarose gel (upper panel) shown is representative of three independent experiments. For RT-qPCR (lower panel), NPM mRNA levels were standardized against RPL32 mRNA levels. The normalized NPM mRNA levels were plotted relatively to the IgG IP condition±s.e.m. of three independent experiments. **P<0.001 (t-test). (b) Schematic representation of the NPM mRNA sequence. The probes covering the NPM 3′-UTR (P1, P2, P1-1 to P1-3) used to generate radiolabelled RNA probes for REMSAs are indicated (black lanes). (c,d,f,h,i) Gel-shift binding assays were performed by incubating 500 ng of purified GST or GST-HuR protein with the radiolabelled cRNA (c) 3′-UTR and 5′-UTR, (d) P1 and P2, (f) P1-1 to P1-3, (h) P1-1, P1-1-mut1 and P1-1-mut2 and (i) P1-1 and P1-1-mut3 probes. These gels are representative of three independent experiments. *shown in panels 4c, f and h indicate the location of shifted complex. (e) BIACORE, a surface plasmon resonance-based biosensor technology, was used for a kinetic binding study between GST-HuR and NPM 3′-UTR, P1 or P2 probes. GST-HuR was captured on a Series S CM5 chip and increased concentrations of NPM cRNA probes, as indicated, were injected over the surface. Injections were performed for 150 s to measure association, followed by a 400 s flow of running buffer to assess dissociation. The association/dissociation ratio of GST-HuR to NPM 3′-UTR is shown in the sensorgram (top left panel). The binding affinity (K_D) of GST-HuR to NPM 3′-UTR (top right panel), P1 or P2 (bottom panels) cRNA probes is also shown. (g) Nucleotide sequence of probe P1-1 showing the thymidine (T) residues that were mutated to cytidine (C) residues (identified by asterisks) to generate the P1-1-mut1, P1-1-mut2 and P1-1-mut3. The sequences highlighted by the dashed boxes as E1 and E2 are the single-strand AU-rich sequences identified using mfold software.