Nucleic Acids Research, 2007, Vol. 35, No. 1 301 Figure 1. The FXR1P isoforms. (A) Schematic representation of the C-terminal region of the three FXR1P isoforms analyzed: Isoe 84 kDa, Isod 78 kDa, Isoa 70 kDa. In the upper part of the figure the alternatively spliced sequences are indicated. A (+) under each amino acid indicates the predicted ability of the sequence to bind RNA accordingly to the algorithm described by Terribilini and coworkers (28). (B) Production of recombinant proteins. Equal amounts of Hist-FMRP, His-FXR1P-Isoe, Isod, Isoa and GST-MSP58 were loaded on a 10% SDS–PAGE gel and revealed by Coomassie blue staining. (Figure 1A). On the other side, it is interesting to underline that the only differences between FXR1P-Isod and Isoe iso- forms are the insertion of 28 amino acid encoded by exon 12 and the presence of 27 amino acid encoded by exon 15 (20,21) (Figure 1A). This 27 amino acid stretch is strongly recognized as a putative RNA binding motif by two different predictive programs available online [http://bindr.gdcb. iastate.edu/RNABindR/main.aspx (27) and http://129.130. 115.77/cgi-bin/bindn.pl (28)], whereas the sequence of exon 12 does not apparently display such properties. The presence or absence of exon 15 raises then the possibility that the 3 iso- forms share different RNA binding abilities. As the tissue distribution of FXR1P isoforms had not been investigated completely, we performed RT–PCR on various RNA samples extracted from cell lines and tissues. FXR1P containing exon 15 RNA was detected at very low level in brain, and in particular in the cerebellum, cortex and hip- pocampus, as well as in the neuroblastoma cell line NG108, together with FXR1P isoforms lacking exon 15 (data not shown). To investigate the G-quartet binding properties of the three FXR1P isoforms, we generated in a bacterial system recom- binant FXR1P isoforms: Isoe, Isod and Isoa (Figure 1A) (3,21), FMRP ISO1 (29) and as a control MSP58, a recently described G-quartet binding protein (19), tagged with His or GST (Figure 1B). In a ﬁlter binding assay, recombinant FMRP protein pro- duced in bacteria displays the same afﬁnity for RNA contain- ing a G-quartet structure as recombinant FMRP produced in an insect cell system (data not shown), conﬁrming that the system of production does not change FMRP afﬁnity for G-quartet RNA, in agreement with studies by Darnell and colleagues (30). Also it has been shown that FMRP acts as a nucleic acids chaperone in low-salt binding conditions (31) and is also able to bind RNA non-speciﬁcally, raising the possibility of introduction of a bias in the assesment of its binding afﬁnities, as already suggested (13,32). Consider- ing the high level of homology that exists between the FXR proteins (4), we reasoned that FXR1P could also display the same properties of aspeciﬁc binding to RNA. As a result, to assess the RNA binding properties of FXR1P isoforms, we used the rigorous and sensitive RNA competition assays, which alleviate the contribution of aspeciﬁc binding (13,25). Using the previously described ﬁlter binding assay (30), we observed that FXR1P-Isod and Isoa isoforms do not bind spe- ciﬁcally G-quartet RNA structure since the amount of bound G-quartet radiolabeled probe is not competed by either the unlabeled G-quartet RNA [N19, corresponding to the portion of the FMR1 transcript containing the G-quartet structure (13)] or another RNA not containing G-quartet structures and not binding FMRP [N8, corresponding to the 30-UTR of the PP2Ac transcript (25)] (Figure 2B). Indeed, at the equi- librium state, the dissociation constant (Kd) is around 5 mM for FXR1P-Isod isoform and 0.8 mM for FXR1P-Isoa. Conversely, FXR1P-Isoe binds G-quartet RNA but with a lower afﬁnity compared to FMRP or MSP58 (Figure 2A). As little as 1 nM of competitor RNA is able to displace 50% of FMRP from G-quartet labeled probe, whereas 10 nM are necessary for FXR1P-Isoe (Figure 2A). When we used as competitor the N8 probe, that does not bind FMRP (25), no binding was observed for all proteins analyzed here (Figure 2B). To conﬁrm that FXR1P-Isoe interaction with G-quartet RNA is speciﬁc for the structure, we per- formed the binding assay either in the presence of K+ or in the presence of Na+. Indeed, FXR1P-Isoe, like FMRP and MSP58 (13,19) is unable to bind G-quartet containing FMR1 RNA in the presence of Na+, a cation destabilizing the G-quartet structure (Figure 2C). This ﬁnding suggests that the effect observed is not due to the recognition of a speciﬁc RNA sequence, but to the G-quartet structure localized in