(A) Kv4.2 (top), histone 1D (H1D) (middle) or Arc mRNA (bottom) (red) in the CA1 region of mouse hippocampus revealed by FISH using anti-sense probe to the 3′UTR together with nuclear marker DAPI (blue); scale bar, 100 μm. Boxed regions are shown at high magnification on the right; scale bar, 30 μm. Arrows point to examples of soma labeled with H1D mRNA (white) or a couple of the numerous cases in which Kv4.2 or Arc mRNA signal colocalizes with YFP-positive (green) dendrites.
(B) Kv4.2 or Arc mRNA (green) in MAP2-positive (magenta) dendrites of mouse hippocampal neurons (DIV14) revealed by FISH using an anti-sense (top) or a sense (middle) probe against Kv4.2-3′UTR, or an anti-sense probe against Arc-3′UTR (bottom); scale bar, 30 μm. Boxed dendrites in left panels are shown at high magnification on the right; scale bar, 10 μm. See Supplemental Figure 1 for more examples.
(C) Kv4.2 mRNA (green) revealed by FISH using an anti-sense probe against Kv4.2-3′UTR, localizes in MAP2-positive (blue) dendrites but not tau1-positive (red) axons in DIV14 mouse hippocampal neurons; scale bar, 20 μm. Boxed region in left panels is shown at high magnification on the right; scale bar, 10 μm.

(A) Schematic depiction of MS2 constructs for visualization of Kv4.2-3′UTR (anti-sense or sense).
(B) Left: Mouse hippocampal neurons (DIV14) co-transfected with MS2-GFP-NLS (green) and MS2BS(6×)-no 3′UTR (top), MS2BS(6×)-Kv4.2-anti-sense 3′UTR (middle) or MS2BS(6×)-Kv4.2-sense 3′UTR (bottom) are shown with MAP2 staining for dendrites (magenta); scale bar, 30 μm. Boxed dendrites in left panels are shown at high magnification on the right; scale bar, 10 μm. Right: Mean GFP intensity of dendrites normalized to that of the nucleus of the same neurons (mean ± S.D.; n is 7, 8 and 8 dendrites each from 4 neurons transfected with MS2BS(6×)-no 3′UTR, Kv4.2-A.S.3′UTR, or Kv4.2-S.3′UTR, respectively. **P<0.01 as compared with MS2BS(6×)-no 3′UTR, Student's unpaired t-test). See Supplemental Figure 2 for sequence similarity between human, rat and mouse Kv4.2-3′UTR, and Supplemental Figure 3 for dendritic targeting of Arc-S.3′UTR as a positive control.

(A) Top: Immunoprecipitation of FMRP from adult mouse brain lysates of WT or fmr1 KO mice. Immunoprecipitated FMRP was immunoblotted with FMRP antibody (top). 10% input (Total) of brain lysates (30 μg) was immunoblotted with FMRP or actin antibody. Bottom: Quantitative measurement of actin mRNA, Kv4.2 mRNA or PSD-95 mRNA co-immunoprecipitated with FMRP from brain lysates of WT or fmr1 KO mice. As described in Supplemental Experimental Procedures, complexes were immunoprecipitated with FMRP antibody and analyzed via qRT-PCR. The input % was calculated by dividing the signal for co-immunoprecipitated mRNA using FMRP antibody by the signal for input (Total) (mean ± S.D.; n=3 for both WT and fmr1 KO mice, *P<0.05 as compared with fmr1 KO, Student's unpaired t-test).
(B) Kv4.2 mRNA (green) revealed by FISH using an anti-sense probe against Kv4.2-3′UTR, co-localizes with FMRP (red) in MAP2-positive (blue) dendrites in DIV14 mouse hippocampal neurons. Merged images of Kv4.2 mRNA and FMRP are shown in yellow and merged images of Kv4.2 mRNA, FMRP and MAP2 are shown in white; scale bar, 20 μm. Boxed dendrites in left panels are shown at high magnification on the right; scale bar, 5 μm.
(C) Time lapse live imaging of neurons transfected with mCherry-FMRP (red), MS2BS(6×)-Kv4.2-sense 3′UTR and MS2-GFP-NLS (green) with indicated time after 100 μM NMDA treatment. Boxed dendrite in the left panel (0 sec; scale bar, 20 μm) is shown at high magnification on the right (0-600 sec; scale bar, 5 μm). Merged images of FMRP (top) and Kv4.2-3′UTR (middle) are shown in yellow (bottom).
(D) Binding assay performed with biotinylated RNA (no RNA, GFP cRNA, Kv4.2-anti-sense 3′UTR or Kv4.2-sense 3′UTR) and mouse brain lysates, as described in Supplemental Experimental Procedures. Complexes were pulled-down with streptavidin-beads and immunoblotted with FMRP antibody (top). 10% input (Total) of brain lysates (30 μg) was immunoblotted with FMRP or actin antibody.
(E) Binding assay performed with biotinylated RNA (no RNA, GFP cRNA, Kv4.2-anti-sense 3′UTR or Kv4.2-sense 3′UTR) and purified GST-mouse full-length FMRP, as described in Supplemental Experimental Procedures. Complexes were pulled-down with streptavidin-beads and immunoblotted with FMRP antibody.
(F) Top: Schematic depiction of FMRP fragments (N-terminus, KH1-KH2 and C-terminus) used for binding assay with biotinylated Kv4.2-3′UTR. Bottom: Binding assay performed with biotinylated Kv4.2-3′UTR and purified GST or GST-FMRP fragments. 10% input of proteins and complexes pulled down with streptavidin-beads were subjected to SDS-PAGE followed by Ponceau staining.
(G) Top: Schematic depiction of Kv4.2-3′UTR fragments (F1, F2, F3, F4 and F5) used for binding assay with purified C-terminal domain of FMRP. Bottom: Binding of purified GST-C-terminal domain of FMRP to biotinylated Kv4.2-3′UTR or some of its fragments. Complexes were pulled-down with streptavidin-beads and immunoblotted with GST antibody. Data shown in (D-G) represent three independent experiments each. See Supplemental Figure 4 for similar experiments with Arc-3′UTR as positive controls and Supplemental Figure 5 for Kv4.2-3′UTR co-immunoprecipitation with FMRP or phospho-S6 but not the RNA-binding protein staufen, with PSD-95-3′UTR as a positive control for co-immunoprecipitation and binding assays.

(A) Relative mRNA level (normalized to GAPDH mRNA) of Kv4.2, PSD-95 or FMRP in hippocampus from WT, fmr1 KO or Kv4.2 KO mice determined by qRT-PCR analysis (WT level was set as 1, mean ± S.D.; n=3 each for WT, fmr1 KO or Kv4.2 KO mice, *P<0.5, **P<0.01 as compared with WT, Student's unpaired t-test).
(B) Left: Co-transfected hippocampal neurons (DIV14) from WT (top) or fmr1 KO (bottom) mice with MS2BS(6×)-Kv4.2-3′UTR and MS2-GFP-NLS (green) are shown with MAP2 staining (magenta); scale bar, 30 μm. Boxed dendrites in left panels are shown at high magnification on the right; scale bar, 10 μm. Right: Mean GFP intensity of dendrites normalized to that of the nucleus in neurons transfected with MS2BS(6×)-Kv4.2-sense 3′UTR from WT or fmr1 KO neurons (mean ± S.D.; n=7 for WT, n=6 for fmr1 KO, P>0.1 as compared with WT, Student's unpaired t-test).
(C) Left: Kv4.2 mRNA in dendrites of hippocampal neurons (DIV14) from WT (top) or fmr1 KO (bottom) mice revealed by FISH using an anti-sense probe against Kv4.2-3′UTR; scale bar, 30 μm. Right: Mean fluorescence intensity of dendritic Kv4.2 mRNA (top) from WT or fmr1 KO hippocampal neurons (mean ± S.D.; n=12 for WT, n=13 for fmr1 KO, P>0.1 as compared with WT, Student's unpaired t-test). Boxed dendrites in left panels are shown at high magnification (bottom); scale bar, 5 μm.

(A) Left: Kv4.2 distribution (black in single images (top); green in merged images (bottom)) in hippocampal slices from 3 week-old or 2 month-old WT (left), fmr1 KO (middle) or Kv4.2 KO (right) mice stained with DAPI (magenta); scale bar, 400 μm. Right: Mean fluorescence intensity of Kv4.2 in CA1 dendritic field of mouse hippocampal slices (marked with *, area quantified: 10184 pixels, mean ± S.D.; n=15 for WT and n=53 for fmr1 KO that are 3 weeks old, n=20 for WT and n=22 for fmr1 KO that are 2 months old, ***P<0.001 as compared with WT of the same age, Student's unpaired t-test).
(B) Left: Kv4.2 levels in hippocampus from WT, fmr1 KO or Kv4.2 KO mice. Mouse hippocampus lysates were immunoblotted with Kv4.2, FRMP or actin antibody. Right: Kv4.2 levels normalized with actin levels (% control to WT, mean ± S.D.; n=4 for both WT and fmr1 KO, **P<0.01 as compared with WT, Student's paired t-test).
(C) Left: Surface biotinylation of hippocampal neurons (DIV14) from WT or fmr1 KO mice, as described in Supplemental Experimental Procedures. Top, 10% input (Total) of cell lysates (30 μg) was immunoblotted with Kv4.2, FMRP or actin antibody. Bottom, biotinylated samples (Surface) were immunoblotted with Kv4.2 or actin antibody. Right: Surface Kv4.2 levels normalized with Total actin levels (% control to WT, mean ± S.D.; n=4 for both WT and fmr1 KO, **P<0.01 as compared with WT, Student's paired t-test).
(D) Left: Kv4.2 surface staining of hippocampal neurons (DIV14) from WT or fmr1 KO mice, as described in Supplemental Experimental Procedures; scale bar, 30 μm. Right: Mean fluorescence intensity of surface Kv4.2 in dendrites (top) from WT or fmr1 KO hippocampal neurons (mean ± S.D.; n=10, 15 and 13 for WT dendrites at 0-50, 50-100 and 100-150 μm from the soma, respectively; n=17, 20 and 18 for fmr1 KO neuronal dendrites at 0-50, 50-100 and 100-150 μm from the soma, respectively, ***P<0.001 as compared with WT, Student's unpaired t-test). Boxed dendrites in left panels are shown at high magnification (bottom); scale bar, 20 μm.
(E) Mean fluorescence intensity of surface Kv4.2 in dendrites (top) from hippocampal neurons transfected with no 3′UTR or Kv4.2-3′UTR (mean ± S.D.; n=7 dendrites for no 3′UTR, n=8 dendrites for Kv4.2-3′UTR, **P<0.01 as compared with no 3′UTR, Student's unpaired t-test). Boxed dendrites in left panels are shown at high magnification (bottom); scale bar, 20 μm.
(F) In vitro translation assay with Renilla luciferase-Kv4.2-3′UTR in presence of GST or GST-FMRP, normalized with firefly luciferase activity (Rluc/Fluc), as described in Supplemental Experimental Procedures (GST control was normalized as 1, mean ± S.D.; n=4, ***P<0.001 as compared with GST, Student's paired t-test).
(G) Left: Live imaging of Dendra-Kv4.2 expressing mouse hippocampal neurons from WT or fmr1 KO mice before, immediately after (0 min), and 60 min after UV exposure, as described in Supplemental Experimental Procedures. Representative grayscale images of green fluorescence in neurons (top); scale bar, 10 μm. Dendrite in orange boxed region with arrow pointing to a single Dendra-Kv4.2 puncta (bottom); scale bar, 5 μm. Right: Normalized newly synthesized Dendra-Kv4.2 (green fluorescence pixel intensity) in individual puncta (mean ± S.D.; n=6 dendrites for both WT and fmr1 KO, **P<0.01 as compared with WT, Student's unpaired t-test).

(A-C) fEPSPs to single pulses of stimulation of Schaffer collaterals were collected from the dendritic field of CA1 neurons for 40 min in WT (A; n=16 slices from 8 animals), fmr1 KO (B; n=27 slices from 3 animals) or fmr1 KO mice in presence of 50 nM HpTx2 (C; n=8 slices from 4 animals). A single train of 5 theta bursts was delivered (arrow, time 0) to the Schaffer collaterals and the group data (mean ± SEM) are expressed as the percentage of mean fEPSP slopes recorded during the baseline (pre-theta burst) period.
(D) Different concentrations of HpTx2 were used to rescue LTP in WT or fmr1 KO mice: 0.3, 1, 3, 10, 30, 50, 100, 300 nM (WT mice: n=3 from 1 animal for 0.3 nM, n=6 from 2 animals each for 1, 3 and 100 nM, n=7 from 3 animals for 10 nM, n=4 from 2 animals for 30 nM, n=5 from 2 animals for 300 nM, fmr1 KO mice: n=3 from 1 animal each for 0.3, 1, 3, 10 and 30 nM, n=8 from 4 animals for 50 nM, n=7 from 4 animals for 100 nM, n=4 from 2 animals for 300 nM. Group data (mean ± SEM) are expressed as the percentage of mean fEPSP slopes recorded during the baseline (pre-theta burst) period.

(A-B) Top: Mouse hippocampal neurons (DIV14-21) were treated with 100 μM NMDA, as described in Experimental Procedures without (A) or with pre-treatment of 20 μM MDL and 25 μM ALLN for 15 min (B). Cell lysates were immunoblotted with Kv4.2 or actin antibody. Bottom: Kv4.2 levels normalized with actin levels (% control to not-treated cells, mean ± S.D.; n=4 for both untreated and pre-treated with MDL+ALLN, **P<0.01 as compared with not-treated cells (black) or 5 min NMDA-treated cells (magenta), Student's paired t-test).
(C) Top: Schematic depiction of luciferase constructs used for luciferase reporter assay. Bottom: Renilla luciferase activity normalized with firefly luciferase activity (Rluc/Fluc) in mouse hippocampal neurons (DIV14) upon NMDA treatment (not-treated cells were normalized as 1, mean ± S.D.; n=3, *P<0.05, **P<0.01 as compared with not-treated cells, Student's paired t-test).
(D) Top: Hippocampal neurons (DIV14-21) from WT or fmr1 KO mice were treated with 100 μM NMDA, as described in Experimental Procedures. Cell lysates were immunoblotted with Kv4.2 or actin antibody. Bottom: Kv4.2 levels normalized with actin levels (% control to WT not-treated cells, mean ± S.D.; n=3, *P<0.05, **P<0.01, ***P<0.001 as compared with not-treated cells (black) or 5 min NMDA-treated cells (magenta), Student's paired t-test).
(E) Top: Schematic depiction of luciferase constructs used for luciferase reporter assay. Bottom: Renilla luciferase activity normalized with firefly luciferase activity (Rluc/Fluc) in hippocampal neurons (DIV14) from WT or fmr1 KO mice, compared between not-treated cells (Control) and 15 min after 5 min NMDA treatment cells (not-treated cells were normalized as 1, mean ± S.D.; n=4, **P<0.01 as compared with not-treated cells (black) or WT (magenta), Student's paired t-test). See Supplemental Figure 6 for lack of NMDAR-induced Kv4.2 up regulation following pre-treatment with calpain inhibitors of neurons from fmr1 KO mice.

(A) Top: Mouse hippocampal neurons (DIV14-21) were treated with 100 μM NMDA for indicated time (0, 2, 5 or 10 min). Cell lysates were immunoblotted with antibody against phospho-FMRP, actin, phospho-mTOR, phospho-S6K, or phospho-S6. Bottom: Phospho-FMRP levels normalized with actin levels (% control to not-treated cells, mean ± S.D.; n=4, ***P<0.001 as compared with not-treated cells, Student's paired t-test).
(B) Top: Mouse hippocampal neurons (DIV14-21) were treated with 100 μM NMDA for 0 or 5 min in the presence of DMSO, 20 nM okadaic acid (loOA), 1 μM okadaic acid (hiOA), or 10 μM cyclosporin A (CysA). Cell lysates were immunoblotted with antibody against phospho-FMRP, actin, phospho-mTOR, phospho-S6K, or phospho-S6. Bottom: Phospho-FMRP levels normalized with actin levels (% control to each not-treated cells, mean ± S.D.; n=3, **P<0.01 as compared with DMSO pre-treated cells, Student's paired t-test).
(C) Top: Renilla luciferase activity normalized with firefly luciferase activity (Rluc/Fluc) in HEK293 cells after transfection with GFP or GFP-tagged FMRP (WT, S499A or S499D) (GFP-transfected cells were normalized as 1, mean ± S.D.; n=4, ***P<0.001 as compared with GFP-transfected cells, Student's paired t -test). Bottom: Cell lysates were immunoblotted with antibody against phospho-FMRP, FMRP or actin. Transfected GFP-FMRPs are only shown in blot.
(D) Top: Mean fluorescence intensity of surface Kv4.2 in dendrites (50-150 μm from soma) of hippocampal neurons (DIV14) from WT or fmr1 KO mice after transfection with GFP or GFP-tagged FMRP (WT, S499A or S499D) (mean ± S.D.; n=8 dendrites for WT (GFP), KO (GFP) and KO (FMRP-WT), n=9 dendrites for KO (FMRP-S499A) and KO (FMRP-S499D), **P<0.01, ***P<0.001 as compared with WT-GFP (black) or KO-GFP (blue), Student's unpaired t-test). Bottom: Kv4.2 surface staining (black in single images (left); magenta in merged images (right)) of hippocampal neurons (DIV14) from WT or fmr1 KO mice, as described in Supplemental Experimental Procedures, after transfection with GFP or GFP-tagged FMRP (WT, S499A or S499D) (green); scale bar, 5 μm. See Supplemental Figure 7 for rapamycin-induced FMRP dephosphorylation and Kv4.2 up regulation, and Supplemental Figure 8 for the block of NMDA-induced FMRP dephosphorylation by PP1 inhibitors.