302 Nucleic Acids Research, 2007, Vol. 35, No. 1 Figure 2. RNA binding properties of FMRP and FXR1P isoforms. (A) Filter binding assay using FMRP, FXR1P-Isoe, Isod, Isoa and MSP58. The RNA probe used is 32P-labeled N19 RNA, and competition was performed using the same unlabeled RNA. (B) The same experiment was repeated using as competitor the N8 RNA sequence, not containaing any G-quartet forming structure. (C) Filter binding assay was repeated with an increasing amount of FMRP and Isoe in the presence of Na+ or K+. (D) GST-pull down was performed as described in Materials and Methods. On the right part of (D), proteins used in GST-pull down assay were revealed by immunoblot. FMRP was detected by monoclonal 1C3 antibody, FXR1P by the monoclonal 3FX antibody. Lane 1: 4 mM GST-FMRP complexed with 1 mM His-FXR1P, Lane 2: 4 mM GST-FMRP complexed with 2 mM His-FXR1P, Lane 3: 4 mM GST-FMRP complexed with 4 mM His-FXR1P. On the left part of (D), proteins used in GST-pull down experiment were revealed by Coomassie stained gel. (E) Competition assay to determine the Kd at the equilibrium state binding FMRP, the heterodimers FMRP/Isoe, FMRP/Isod, FMRP/Isoa and the complex FMRP/MSP58, with the 32P-labeled N19 probe and competed with unlabeled N19. (F) The same experiment described in (E) was repeated using the N8 RNA as unlabeled competitor. the assayed RNAs (13). In addition, we repeated the same analysis by competing the binding of 32P-labeled N19 probe with the G-quartet forming RNA structures obtained from the 50-UTR of PP2Ac (that contains four G-quartet forming structures) (25) and obtained the same results (data not shown) that are described in Figure 2A using N19 RNA competition. Since FXR1P isoforms display different G-quartet binding speciﬁcity, we asked whether they would affect differently FMRP binding afﬁnity to G-quartet. First, we veriﬁed the