2 mRNA coimmunoprecipitated

with FMRP from the adult mouse brain lysate (Figure 3A), similar to the coimmunoprecipitation of FMRP with PSD-95 mRNA, another target of FMRP. Finally, we monitored concerted movements of FMRP and Kv4.2-3′UTR by live imaging of neurons expressing MS2-GFP-NLS and MS2BS(6X)-Kv4.2-S.3′UTR together with fluorescently tagged FMRP following NMDAR activation, which enhanced the movement of these granules (Figure 3C). Taken together, these findings indicate that FMRP is associated with click here Kv4.2 mRNA in neuronal dendrites. We then tested for binding of FMRP to the 3′UTR of Kv4.2 mRNA, because in silico analysis of this region has revealed the presence of U-rich stretches (Figure S2), a sequence motif for RNA binding to FMRP (Chen et al., 2003). By using streptavidin-beads to pull down proteins from brain lysates bound to biotinylated Kv4.2-3′UTR, we found FMRP binding of Kv4.2-S.3′UTR (Figure 3D) at a level comparable to that of Arc-3′UTR or PSD-95-3′UTR (Figures S4B and S5A). This binding is specific to FMRP because Kv4.2-3′UTR

showed no association with the RNA-binding protein Staufen or non-RNA binding proteins SCH 900776 such as mTOR, dynamin 1, and actin (Figure S5A). Furthermore, the binding is direct as evident from the interaction between bacterially expressed and purified FMRP and Kv4.2-S.3′UTR, at a level comparable to the interaction between FMRP and PSD-95-3′UTR (Figure 3E; Figure S5B). This binding is specific because FMRP binds Kv4.2-3′UTR but not GFP mRNA or Kv4.2-A.S.3′UTR (Figures 3D and 3E). Next, we examined the three domains Cytidine deaminase of FMRP individually. Only the C-terminal domain of FMRP was specifically pulled-down with Kv4.2-3′UTR (Figure 3F). This domain contains an RGG box known to have an affinity for mRNAs. We then tested five RNA fragments that together encompass the entire 3′UTR of the mouse Kv4.2 mRNA, and found only

fragment 2 and fragment 5 that contain U-rich sequences associated with the purified FMRP C-terminal domain (Figure 3G). Notably, fragment 2 includes an evolutionarily conserved U-rich sequence (Figure S2). Taken together, these studies show that the direct interaction between FMRP and Kv4.2-3′UTR is likely evolutionarily conserved. We found the Kv4.2 mRNA level in the hippocampus of fmr1 KO mice was similar to that in wild-type (WT) mice ( Figure 4A). We confirmed the gene targeting using primers that amplify exon 5 (or exon 1) of the fmr1 (or Kv4.2) gene that is interrupted by the neomycin resistance selection marker gene in the fmr1 (or Kv4.2) KO mice ( Figure 4A); using other primers we found that these KO mice have some remnant, genetically altered, transcripts. Using the MS2 system to track the subcellular localization of MS2BS(6X)-Kv4.2-S.3′UTR in hippocampal neurons with or without FMRP, we found similar dendritic targeting ( Figure 4B), indicating that FMRP is not required for dendritic targeting of Kv4.2-3′UTR.