304 Nucleic Acids Research, 2007, Vol. 35, No. 1 Figure 4. Dissociation rate of FMRP, Isoe, MSP58 and the two complexes FMRP/Isoe and FMRP/MSP58. (A) Each protein was mixed with 32P-labeled N19 RNA probe for 10 min on ice and then an equal amount of unlabeled N19 RNA was added as competitor to each reaction, which was then filtered after a precise time lapse of 10, 30, 60, 120, 180, 240 and 300 min. The radioactivity retained on the filter was evaluated. (B) The same experiment described in (A) was repeated using unlabeled N8 RNA as competitor. (C) The same experiment described in (A) was performed using the protein complexes FMRP/Isoe and FMRP/MSP58. (D) The same experiment described in (C) was performed using the cold N8 RNA as competitor. of proteins interacting with FMRP has been described, most of them being RNA binding proteins (19,33). In addition, several hundreds of mRNAs have been described as putative targets of FMRP (12,26), however the functional signiﬁcance of most of the multiple interactions established by FMRP is still elusive (12,34). A widely accepted hypothesis proposes that FMRP can transport mRNA in mRNPs shuttling between structures where RNA translation is repressed and polyribo- somes (2). In the absence of FMRP, the equilibrium in mRNP normally containing FMRP is perturbed, resulting in the deregulation of the expression and localization of a subset of its target mRNAs (12,26). Based on these considerations, we reasoned that RNA binding proteins belonging to the same mRNP complex, FMRP and its interacting proteins may enter in contact with the same mRNAs and decided to test the ability of FXR1P to bind the same mRNA targets and to inﬂuence its afﬁnity for them. Up to date, the functions of FXR1P (or FXR2P) were inferred to be, by homology and analogy, similar to that of FMRP (1). Here we propose that it is not the case, at least for FXR1P. First, we tested FXR1P afﬁnity for G-quartet forming RNA structures. It is surprising that among the three isoforms analyzed only one, the Isoe is able to bind a G-quartet RNA forming structure, present in a large amount of putative target RNAs of FMRP (26). The three FXR1P isoforms share the same RGG box domain. However, it has been reported that, even though a peptide corresponding to the RGG box of FMRP binds speciﬁcally G-quartet forming RNA (30), the corresponding peptide of the RGG box of FXR1P does not (35), strongly suggesting that the RGG box of FXR1P is not sufﬁcient per se to bind the G-quartet structure. The only difference between the two isoforms Isoe and Isod are two short sequences of 28 (exon 12) and 27 amino acid (exon 15) (cf. Figure 1A). Only this latter amino acids stretch appears to have putative RNA binding properties and is encountered solely in the Isoe isoform able to bind speciﬁc- ally the G-quartet structure. This 27 amino acid stretch encoded by FXR1P exon 15 being in close proximity to the RGG box of FXR1P encoded by sequences of exon 14 (Figure 1A), it may contribute to the binding to G-quartet mRNA structures together with the RGG box. Alternatively, the presence of this additional sequence in the FXR1P-Isoe, as compared to the other shorter isoforms, may alter the struc- ture of the C-terminal portion of FXR1P, thereby allowing the binding. In a similar way, a different afﬁnity for RNA was also shown for different FMRP isoforms. Indeed, ISO18, a minor isoform of FMRP lacking a small portion of exon 17 (29), is still able to bind G-quartet RNA (36),