Nucleic Acids Research, 2007, Vol. 35, No. 1 303 Figure 3. Association rate of FMRP, Isoe and MSP58. (A) Each protein was mixed with 32P-labeled N19 RNA probe for a time lapse of 10, 30, 60, 120, 180, 240 and 300 min and then each reaction was filtered and the amount of retained radioactivity evaluated. (B) The same experiment described in (A) was repeated with the complex FMRP/MSP58 and FMRP/Isoe as indicated in the figure. amounts of FXR1P and FMRP that integrate the heterodimer complex. To this purpose, we performed GST-pull down experiments by mixing 4 mM of GST-FMRP with increasing amounts (1, 2, 4 mM ) of His-FXR1P-Isoe, Isod or Isoa (In Figure 2D, the results are shown only for interaction bet- ween FMRP and FXR1P-Isoe). The beads were then treated with glutathione and the eluted proteins were revealed by immunoblot using the two monoclonal antibodies 1C3 (24) and 3FX (21) recognizing FMRP and FXR1P, respectively. As shown in Figure 2D, the ratio of released FXR1P and FMRP is around 1:1 when mixed in stochiometric amounts. This result shows that when the two proteins are mixed in vitro their association is dose-dependent and, on the other side, also shows that our results are not due to an unbalanced ratio of the two interacting proteins in the FXR heterodimers. Subsequently, we tested the ability of the FXR1P-Isoe/ FMRP heterodimer to bind G-quartet RNA. Indeed, the FXR1P-Isoe/FMRP complex binds G-quartet RNA with a comparable afﬁnity as the FMRP homodimer at different concentrations of competitor RNA (Figure 2E). Surprisingly, FXR1P-Isod and Isoa inhibit FMRP binding to G-quartet RNA when these form a heterodimer with the latter protein (Figure 2E). As a control, MSP58 protein, that binds G-quartet RNA in a speciﬁc manner (19), was used (Figure 2E), and its binding to FMRP leads to the same results as when FMRP is complexed to FXR1P-Isoe. When we used the N8 probe as a negative control, no displacement of the equilibrium was observed, as shown in Figure 2F. In view of these results, we decided to better dissect the dynamics of FXR1P/G-quartet RNA and FXR1P/FMRP/ G-quartet RNA interactions. We evaluated the velocity of interaction of the two FXR proteins with G-quartet RNA. For this purpose 1 pmol of each protein was mixed with 5 fmol of labeled N19 RNA. At different time points (10, 30, 60, 120, 240 or 300 min), the assay was stopped and the amount of radioactivity bound by the proteins evaluated by the ﬁlter binding assay. The time necessary for FMRP/FXR1P-Isoe heterodimer to bind the half amount of total bound RNA ligand was estimated to be 9.93 min, which is lower than the time employed by FMRP or FXR1P homodimers (33.65 and 19.04 min, respectively) to bind the same amount of RNA probe (Figure 3A and B ). This result indicates that the Kon for the heterodimer is higher than the Kon for both homodimers. Conversely, the presence of MSP58 complexed with FMRP did not inﬂuence its binding to G-quartet RNA. We then investigated the kinetics of FXR1P-Isoe and FMRP dissociation from the G-quartet RNA structure, when both bind to it as homodimers or as heterodimers. FMRP was mixed with 32P-labeled N19 RNA and the com- plex was allowed to form for 10 min on ice. Then 1 mM of cold RNA [N19 or N8, as a negative control (25)] was added, the reaction was stopped after 10, 30, 60, 120, 240 or 300 min and the amounts of retained radioactivity evalu- ated by ﬁlter binding assay. In this experiment, it is interest- ing to observe (Figure 4A) that the FXR1P-Isoe homodimer releases 50% of total bound RNA after only 15 min. As expected from its higher afﬁnity, the FMRP homodimer releases the same amount of bound RNA after a longer lapse of time, around 45 min. The negative controls are shown in Figure 4B. In this case the binding of recombinant proteins to G-quartet containing RNA was competed using the N8 probe. Indeed, only 20–25% of the binding is competed after more than 4 h of incubation. Finally, the heterodimer FXR1P-Isoe/FMRP and the complex MSP58/FMRP were analyzed. The effect of the heterodimer was dramatic: 50% of the labeled RNA was released after 5 min and 65% of labeled RNA was released after 10 min (Figure 4C), while the interaction with MSP58 does not change signiﬁcantly the dynamics of the interaction between the G-quartet RNA and FMRP. In Figure 4D the negative controls are shown, conﬁrming the speciﬁcity of the action described in Figure 4C. This data shows that formation of the FXR1P-Isoe/FMRP heterodimer increases the dynamics of protein–RNA interaction, favoring the release of bound mRNA. DISCUSSION FMRP is a component of multimolecular complexes involved in different steps of mRNA metabolism (2,12). A growing list