300 Nucleic Acids Research, 2007, Vol. 35, No. 1 spliced, with the possibility to generate upto 15 isoforms (20), see also www.ncbi.nlm.nih.gov/IEB/Research/Acembly/. Of notice, some of these isoforms are differentially expressed in various tissues (21). Up to date, the ability of full-length FXR1P and FXR2P to bind a G-quartet RNA structure in a speciﬁc manner has not been reported. We analyzed here the RNA binding properties of the three most abundantly expressed FXR1P isoforms and show that they have different afﬁnities for the G-quartet RNA structure. Since all protein members of the FXR family are able to heterodimerize with FMRP, they are believed to act together (4). In the present study we determined that, when complexed to FMRP, FXR1P isoforms can modulate its afﬁnity for G-quartet RNA and also the dynamics of this complex. Our data demonstrate that FXR1P has a synergistic molecular function with FMRP rather than a redundant role. MATERIALS AND METHODS Purification of recombinant proteins Glutathione S-transferase (GST)-FMRP produced in the baculovirus system was puriﬁed as described previously (22). pET21a/FMRP (ISO1) vector was described previously (23). To construct pGex-4T-1/FMRP, ISO1 cDNA was excised from pTL1/FMRP ISO1 and subcloned into the EcoRI/NotI sites of pGex-4T-1 (Amersham). To construct pET21a/FXR1P, Isoe, Isod and Isoa isoforms were ampliﬁed by PCR using the primers (Eurogentec): EcoRI forward-50- GGCGAATTCATGGCGGACGTGACGGTG-30; XhoI reverse- 50-GCCCTCGAGTTATGAAACACCATTCAGGAC -30, the PCR consisted of 1 cycle at 94 C for 4 min, 30 cycles of three steps each, 94 C for 30 s followed by 60 C for 30 s and 68 C for 2 min using the Pfx polymerase (Invitrogen). PCR fragments were puriﬁed, digested and cloned into the EcoRI/XhoI sites of pET21a (Novagen). The sequences of the cDNAs corresponding to the different FXR1P isoforms were veriﬁed by sequencing. The proteins were produced in bacteria and puriﬁed following the manufacturer’s protocol. GST- MSP58 was produced and puriﬁed as described previously (19). GST-pull down GST-pull down assays were performed as described previ- ously (22). Brieﬂy, an increasing amount of recombinant His-FXR1P (1, 2 or 4 mM) was mixed with 4 mM of GST- FMRP. Pull down assays were carried out in the following buffer: [50 mM Tris–HCl (pH 7.4) at 4 C, 1 mM MgCl2, 1 mM EDTA, 150 mM KCl, 1 mM DTT], as described (22). After washing with the same buffer, the proteins bound to the beads and their interactors were eluted using 30 mM glu- tathione and separated by electrophoresis on 8% SDS– polyacrylamide gels. FMRP was visualized by immunoblot using the 1C3 monoclonal antibody (24), FXR1P was revealed by the 3FX monoclonal antibody (21). The proteins were also visualized on gel by Coomassie staining. RNA binding assays The different RNA fragments used in this study, N19 [RNA sequence derived from FMR1 cDNA and containing a G-quartet forming structure (13)] and N8 [RNA sequence not containing G-quartet structures and corresponding to the 30-untranslated region (30-UTR) of PP2Ac (25)], were cloned in pTL1 plasmid. For ﬁlter binding assay, pTL1 plasmids linearized with PstI were in vitro transcripbed with T7 RNA polymerase (Promega) (13). The RNAs were puriﬁed using the NucAway Spin columns (Ambion). RNAs were then ethanol precipitated and resuspended in a appropriate buffer. For binding experiments, N19 was labeled co- transcriptionally by incorporation of [a-32P]ATP. Labeled RNAs were puriﬁed on a 1% low-melting agarose gel (Ambion). Labeled RNAs (80 000 c.p.m., 5 fmol) were rena- tured for 10 min at 40 C in 4 ml of binding buffer [50 mM Tris–HCl (pH 7.4) at 4 C, 1 mM MgCl2, 1 mM EDTA, 150 mM KCl, 1 mM DTT] in the presence of 8 U RNasin (Invitrogen), 0, 1 mg of Escherichia coli total tRNA and 0.01% BSA. An increasing amount of protein was then added to the RNA. RNA–protein complexes were formed for 10 min on ice. After incubation, binding solutions were passed through MF-membrane ﬁlters (0.45 HA, Millipore) and washed with 2 ml binding buffer. Filters were air dried and the amount of radioactivity was measured by Cerenkov counting. Data were plotted as percentage of total RNA bound versus the protein concentration. Competition experi- ments to determine the relative binding strength of the differ- ent proteins to G-quartet RNA were carried out using labeled N19 RNA incubated with 1 pmol of protein in the presence of increasing concentrations of unlabeled competitors. FMRP was used as an internal positive control. For association rate determination, 5 fmol of labeled N19 were incubated with 1 pmol of the appropriate protein in the binding buffer between 10 and 300 min on ice. For dissociation rate deter- mination, 5 fmol of labeled N19 were incubated with 1 pmol of the appropriate protein in the binding buffer for 10 min on ice, 10 6 M of competitor RNA (N19 or N8) were then added to the mixture and incubated between 10 and 300 min. Each binding curve is the result of at least three independent experiments performed with three replicates for each binding point. All data obtained for the different experi- ments of RNA binding, calculating the standard deviation for each binding point, are shown in Supplementary Data. All the values and curves were analyzed using the PRISM Graphpad version 4 Software. RESULTS Our ﬁrst aim was to assess whether FXR1P is able to bind G-quartet RNA structure, which is considered to be a frequent structure recognized by FMRP and present in many of its mRNA targets (12,13,26). Due to extensive alternative splicing of FXR1 mRNA, at least seven isoforms of FXR1P are differentially expressed in various tissues (20). We decided to study the RNA binding properties of three FXR1P isoforms: Isod and Isoa (Figure 1), the two isoforms most highly expressed in brain (3), and Isoe (Figure 1) that is a FXR1P isoform highly expressed during myogenesis and in adult cardiac and skeletal muscle (21). The FXR1P-Isod and Isoa isoforms both lack exon 12 and 15 and only differ in their C-terminus due to the choice of a different splicing acceptor site in the mRNA of the FXR1P-Isoa isoform, result- ing in a frameshift that induces an early stop codon