Supplementary Information for - PNAS

Supplementary Information for Rapid, Experience-Dependent Translation of Neurogranin Enables Memory Encoding Kendrick J. Jonesa, Sebastian Templeta,b, Khaled Zemouraa, Bozena Kuzniewskaf, Franciso X. Penaa,b, Hongik Hwanga,c,1, Ding J. Leia,d, Henny Haensgena, Shannon Nguyene, Christopher Saenza,b, Michael Lewise, Magdalena Dziembowskaf and Weifeng Xua,b,2 a

Picower Institute for Learning and Memory, bDepartment of Brain and Cognitive

Sciences, cDepartment of Chemistry, dDepartment of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, USA, eStanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, MA 02142, USA, fCentre of New Technologies, University of Warsaw, S. Banacha 2c, 02-097 Warsaw, Poland Weifeng Xu Email: [email protected]

1 www.pnas.org/cgi/doi/10.1073/pnas.1716750115

This PDF file includes: Supplementary text Figs. S1 to S6 Tables S1 to S2 References for SI reference citations Other supplementary materials for this manuscript include the following: Datasets S1

2

SI Materials and Methods Commercially Available Resources Supplemental Index, Table S2 Animals All animals were maintained in a vivarium with a light/dark cycle (7:00 a.m.-7:00 p.m.). Animal care and handling were performed according to NIH guidelines and with the approval of the Massachusetts Institute of Technology (MIT) institutional animal care and use committee (IACUC) and Division of Comparative Medicine (DCM). AAV Expression Constructs The 1.3 kB CaMK promoter was subcloned from pLenti-CaMKIIa-hChR2-EYFP-WPRE (64) in an adeno-associated viral vectors (AAV) with AAV2 ITRs (65). The Ng 3’-UTR, Gapdh 3’-UTR and truncation Ng 389-577 were PCR amplified and cloned into an GFP- expressing AAV vector under the control of the CaMK promoter. Neurogranin cDNA was PCR amplified from the human neurogranin cDNA from ATCC (MGC-3856), and Ng and NgΔIQ deletion construct were cloned into an AAV expression vector under the CaMK promoter in frame with eGFP. Detailed primer sequence and cloning strategy in supplemental methods. Target sequences for Luciferase (CGTACGCGGAATACTTCGA) and for Fmr1 (66) (GTGATGAAGTTGAGGTTTA) were used for generating AAV vectors of shLuc and shFmr1 under the H1 promoter(65, 67). The same CaMKpromoter-Ng- eGFP cassette from above was used to generated the rescue AAV constrcuts shLuc-Ng- eGFP and shFmr1-Ng-eGFP.

3

Viral Production AAV 2/9 serotype AAV vectors were produced as previously described (67-69). Viral Injection Male C57BL/6Ncrl mice were prepared for surgery using standard procedures (67) in accordance with NIH guidelines and with the approval of the MIT IACUC and DCM. The coordinates for the dentate gyrus are anterior-posterior (AP), −2.2 mm;; medial-lateral (ML), ±1.2 mm from bregma;; dorsal-ventral (DV), −1.6 mm below the dura. Drugs Anisomycin (150 mg/kg IP) was dissolved in 0.1 M HCl and pH adjusted with phosphate buffered saline and injected at 10 ml/kg. Bicuculline methobromide was dissolved in ddH2O at 40 mM and used at 40 µM. Actinomycin D was dissolved in ddH2O at 5mM and used at 5 µM. Cycloheximide was dissolved in methanol at 100 mM (for cell culture experiments) and used at 100 µM or 100 mg/ml and used at 100 µg/ml (for polyribosomes). Behavioral Analysis Context memory test with pre-exposure (70): C57BL/6Ncrl 7-9 weeks-old male mice were habituated to the room for 4 hours, 48 and 24 hours prior to experiments. 19 cm x19 cm with 16 stainless steel rods as a floor (Coulbourn Instrumnents) were used as the context. The chamber was cleaned with Quatricide before and between each subject. The chamber had a light ethanol scent. Animals were exposed to the context for the indicated time. 24 hours later, animals were placed in the chamber, given an immediate shock (0.7 mA, 2 sec) through the floor rods with a shock generator (Coulbourn Instrumnents) and removed from the chamber after a total of 1 minute. 30

4

minutes later, animals were re-exposed to the chamber for 3 minutes to assay freezing. At least two cohorts of the animals in each experiment were blinded to the experimenter. Animal IDs were blinded for data analyses. The Fmr1 KO mouse line was backcrossed >10 generations with C57BL/6Ncrl. 7-9 week old knockout and wild type littermates were tested in the same manner as above. Novel Context Exposure: Animals were individually housed 72 hours prior to the experiment. 48 and 24 hours prior to experiment animals were habituated to the room for 4 hours. The novel context was either the contextual fear conditioning chamber described above or a 40.6 x 40.6 x 30.5 cm plexiglass chamber. Animals were allowed to explore the chamber for 8 minutes and returned to the home cage for 7 minutes. Open Field: Locomotion and center crossings were measured in open field chambers (41.9 x 40.6  x 30.5  cm;; AccuScan Instruments). Activity was detected by infrared beam breaks and recorded automatically by VersaMax software (AccuScan Instruments). Distance traveled (cm) data were automatically binned in 5-minute intervals and total distance traveled. Elevated Plus Maze: Mice were tested on a 4 arm elevated plus maze. Closed arms were 25 x 5 x 5 and open arms were 25 x 5 x 0 cm. Animals were placed in the center of the maze and allowed to explore for 5 min. Time spent in each arm and number of entries into the open arms were quantified using Ethovision (Noldus) software. Polyribosome Enrichment and Real Time RT-PCR Mice were rapidly decapitated and submerged in liquid nitrogen for 4 seconds to rapidly cool brain tissue. Hippocampi were dissected on ice within 90 seconds, and homogenized, and polyribosome enrichment was performed as described (Fig. 2B) (71, 72).

5

RNA was quantified with a NanoDrop and reverse transcribed using Superscript VILO (Invitrogen) using 25 – 50 ng of RNA. Samples with yield less than 100 ng RNA or produced 260/280 absorbance ratios less than 1.8 were discarded from future analyses. Real time PCR was performed using Ssofast Evagreen supermix (BioRad) on a C1000 thermal cycler with the CFX96 detection system (BioRad). Fold change for each experimental cDNA was normalized to GAPDH cDNA within each sample. Relative fold change between novel and control samples was then calculated using 2-ΔΔCt. Ribosomal 18s enrichment between samples was calculated using absolute comparisons of the r18s Ct values. Samples with 18s enrichment values less than 2 were discarded from future analyses. Negative controls lacking the RT enzyme or lacking a template strand were run in parallel for all samples and primers. qRT-PCR primer sequences and references are given in Supplementary Table S1. 3-5 different primer pairs, designed in- house and from multiple databases, for each gene candidate were tested and validated to identify one primer set with the highest specificity and amplification efficiency. Western Blotting Animals were sacrificed by cervical dislocation in a separate room from the behavioral room. Brain regions were rapidly dissected on ice and flash frozen in liquid nitrogen. Brain tissues were homogenized in (mM) 5 HEPES pH7.4, 2 EDTA, 2 EGTA, and 1% SDS and boiled for 10 minutes. 25 µg total protein lysates were loaded onto a 4-12% bis-tris gel (Invitrogen). Dissociated neuronal cultures were lysed in 2 x sample buffer with BME, boiled 10 min and analyzed by western blot. SDS-PAGE gels were transferred to 0.2 µm pore PVDF membranes (Millipore) at 45 V for 60 minutes at 4°C. Signals from infrared secondary antibodies (Licor) were detected on an Odyssey scanner (Licor) and quantified using ImageJ.

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Cell Culture Dissociated cortical cultures were prepared from newborn C57BL/6Ncrl as previously described (73). Proximity Ligase Assay In situ PLA was performed using the DuoLink II kit (Sigma) according to the instructions of the manufacturer. The primary neurons were fixed using 4% paraformaldehyde in PBS (PFA) and later permeabilized using 0.2% Triton X-100, subjected to rabbit anti-Ng or anti-CaM and mouse anti-puromycin incubation and subsequent PLA reaction. Coverslips were mounted with fluorescence mounting medium (Dako) to subject to confocal microscopy. RNA Pull-Down Assay The RNA pull down assay was based on (74). UTR constructs were cloned into pBluescript SK(-) and transcribed using T7 polymerase (Ambion) in the presence of Biotin-16 UTP. 5 µg of in vitro transcribed RNAs was incubated and attached to 30 µl streptavidin dynabeads (Invitrogen) in incubation buffer (10 mM Tris HCl pH 7.4, 1 mM EDTA, 2 M NaCl) for 20 min at room temperature. Hippocampi were homogenized in 3 ml ice-cold extraction buffer (NaCl 100 mM, MgCl2 10 mM, TrisHCl pH 7.5, 10 mM, Triton X100 1%, DTT 1 mM, protease inhibitor cocktail 10 µl/ml, phosphatase inhibitor cocktails 10 µl/ml, RNaseOUT 40U/mL) Beads were incubated overnight with rotation at 4°C. Beads were washed and proteins were eluted in 1% SDS and separated on SDS- PAGE gel (Invitrogen) for either western blotting or Mass Spec (MIT Whitehead Proteomics Core). Mass spec was performed independently two times and proteins found in both samples were marked in bold (Supplemental Index, Dataset S1).

7

Immunohistochemistry C57BL/6Ncrl mice (8 weeks old) were anesthetized with Isofluorane (Piramal Healthcare) and transcardially perfused with 4% PFA. After post-fixation, brains were sectioned (45 µm) on a vibratome (Leica). Slices were blocked and permeablized in 10% horse serum, 1% goat serum, 1% BSA, 0.5 % TritonX-100 in PBS, incubated overnight at 4°C with anti-Ng (1:500) and anti-synaptophysin (Sigma, 1:1000), then with secondary antibody goat anti-rabbit 488 and goat anti-mouse 546 (1: 5000, Invitrogen), mounted in Prolong Gold anti-fade reagent (Thermo-Fisher Scientific) with PBS washes in between steps. Sections were imaged with laser scanning confocal microscopy, using identical gain settings and laser intensities between naive and novel context exposed animals. The whole brain coronal sections were taken with a 5x objective (Zeiss 710) for 5 optical sections spaced by 10 µm. Images of the DG and CA regions were taken with a 63 x oil immerse objective (NA 1.40;; WD 0.17 mm, Zeiss 810), for 10 to 15 optical sections spaced by 0.8 µm. at a resolution of 1024 x 1024 pixels and processed in Imaris and Adobe Photoshop. Polyribosome

profiling

and

RNA

quantification

from

stimulated

synaptoneurosomes Procedures were described previously (75-77). Synaptoneurosomes (SN) were prepared from 1- to 2-month-old WT and Fmr1 KO male mice on FVB background. Hippocampi and cortices were dissected and homogenized in cold Krebs buffer (2.5 mM CaCl2, 1.18 mM KH2PO4, 118.5 mM NaCl, 24.9 mM NaHCO3, 1.18 mM MgSO4, 3.8 mM MgCl2, 212.7 mM glucose;; pH7.4 set with carbogen) supplemented with 1× protease inhibitor cocktail EDTA-free (Roche) and 120 U/ml RNase Inhibitor (RiboLock, ThermoScientific). Homogenates were passed through a series of nylon mesh filters consecutively: 100, 60, 30 and 10 µm (Millipore), centrifuged at 1000 X g for 15 min at 4°C. Pellet was washed

8

and resuspended in Krebs buffer with protease and RNase inhibitors. Next, synaptoneurosomes were prewarmed for 5 min at 37°C and stimulated with a pulse of 50 µM NMDA and 10 µM glutamate for 30 sec, then APV (120 µM) was added and synaptoneurosomes were further incubated for 20 min at 37°C (Scheetz 2000). Unstimulated samples, kept on ice with 200 µg/ml cycloheximide, were used as controls. After the stimulation synaptoneurosomes were centrifuged at 1000 x g for 15 min at 4°C and the pellet was lysed in 1 ml of lysis buffer (20 mM Tris-HCl pH 7.4, 2 mM DTT, 125 mM NaCl, 10 mM MgCl2, 200 µg/ml cycloheximide, 120 U/ml RNase inhibitor, 1 × protease inhibitor cocktail and 1.5% IGEPAL CA-630 (Sigma Aldrich)). After centrifugation at 20,000 x g for 15 min at 4°C, the supernatant was loaded on a 10–50% linear density gradient of sucrose and ultracentrifuged at 38,000 x rpm for 2 h (TH641 rotor;; Sorvall WX Ultra Series Centrifuge;; Thermo Scientific). Gradients were collected using BR-188 Density Gradient Fractionation System (Brandel). Based on the UV profile fractions were combined into three pools: monosome, light polyribosomes and heavy polyribosomes. Before the RNA extraction, external spike-in control mRNA (an in vitro transcribed fragment of A. thaliana LSm gene, 10 ng) was added to each of the fractions to control the efficiency of RNA precipitation. RNA in each fraction was supplemented with linear polyacrylamide (20 µg/ml) and precipitated with 1:10 volume of 3 M sodium acetate, pH 5.2 and 1 volume of isopropanol. After proteinase K digestion RNA was isolated using phenol/chloroform extraction method. RNA pellets were dissolved in 30 µl of RNase-free water. Equal volume from each RNA sample (6 µl) was reverse transcribed using random primers (GeneON;; #S300;; 200 ng/RT reaction) and SuperScript IV Reverse Transcriptase (Thermo Fisher). Next, the abundance of studied mRNAs in different

9

polysomal fractions was analyzed by qRT-PCR using Light Cycler 480 Probes Master Mix (Roche) in a LightCycler480 (Roche). The cDNA was diluted 5 × with H2O and 4 µl of each cDNA sample was amplified using a set of custom sequence-specific primers and TaqMan MGB probes in a final reaction volume of 15 µl. Following TaqMan Gene Expression Assays (Thermo Fisher) were used: i) Nrgn, Neurogranin (Ng);; (Mus musculus);;

Mm01178296_g1;;

ii)

Gap43,

Neuromodulin,

(Mus

musculus);;

Mm00500404_m1;; iii) Calm1, Calmodulin, (Mus musculus);; Mm01336281_g1;; iv) AT3G14080,

U6

snRNA-associated

Sm-like

protein

LSm1

(Arabidopsis);;

At02174019_g1. Relative mRNA levels in different fractions were determined using the ∆Ct (where Ct is threshold cycle) relative quantification method and presented as the % of mRNA in each fraction. Values were normalized to LSm spike-in control. Statistical Analysis Statistical analysis was performed using Prism (Graphpad). Group differences were determined using either one-way ANOVA or two-way ANOVA with appropriate post-hoc test. One sample t-test was used for comparison of a group of data with a fixed value. Significance threshold was set at p = 0.05.

10

Figure S1. Sufficient pre-exposure to a context allows associative learning (associated with Figure 1). (A). Schematic for the contextual memory test. (B). Quantification of freezing percentage of animals exposed to a novel context for 0 (n = 10), 2 (n = 14), or 8 (n = 15) minutes. One-way ANOVA, F(2,36) = 26.79, p < 0.0001, followed by Tukey’s test. **** p < 0.0001.

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Figure S2. Nrgn mRNA Levels are increased in the polyribosomal fraction but not in the total homogenate, unlike the IEGs, following novel context exposure (associated with Figure 2). (A). Quantification of relative levels (Fold Change Nov/Ctrl) of IEG mRNA candidates in the hippocampal ribosome enriched fraction from novel context-exposed (Nov) animals to those from control animals (Ctrl). Colored symbols, individual data points;; colors, different cohorts of animals, n = 7 out of three cohorts;; mean ± S.E.M is represented in solid black lines in each data column. One-way ANOVA, F(5,36) = 15.22, p < 0.0001, followed by Dunnett’s test, compared to Tubb3 ratio as a control. **** p < 0.0001. (B). Quantification of relative levels (Fold Change Nov/Ctrl) of IEG mRNA candidates and selected targets in the hippocampal input from novel context-exposed (Nov) animals to those from control animals (Ctrl). Data presentation as in (A). Mean ± S.E.M is represented in solid black lines in each data column. One-way ANOVA, F(7,48) = 15.62, p < 0.0001, followed by Dunnett’s test, compared to Tubb3 ratio as a control. **** p < 0.0001.

12

Figure S3. Ng protein levels are correlated with the Nrgn mRNA levels in the polyribosome-enriched fraction (Associated with Figure 2). (A). Representative western blot images of Ng in hippocampal lysate from control (Ctrl) and novel context exposed (Nov) animals. (B). Correlation of fold change in protein levels (Nov/Ctrl) and the fold change in Nrgn mRNA in polyribosome enriched fraction (Nov/Ctrl). Each colored symbols represents an individual data point from one pair of comparison. Different cohorts of animals were color coded.

13

H 7500 5000 2500 0

N

10 5 0

I

15

% Center Time

10000

gN ∆IQ g3 ’U GF T P NR N g-G g3 F ’U PTR

Ng-eGFP-Ng3’UTR

G

10 5 0

N gN ∆IQ g3 ’U GF TR P N N g-G g3 F ’U PTR

Ng∆IQ-eGFP-Ng3’UTR

Total Dist (cm)

F

0

G eGF 3’ P U TR e Ng G 3’U FP TR -

G eGF 3’ P U TR Ng eG 3’U FP TR -

0

5

% Time in Open Arm

2500

10

15 10 5 0


5000

15

J 15 10 5 0

N % Time in Open Arm gN ∆IQ g3 ’U GF T P NR N g-G g3 F ’U PTR

7500

15

% Center Time

eGFP-Ng3’UTR

Total Dist (cm)

10000

E

# of Open Arm Entries

eGFP-G3’UTR

D


C

g# of Open Arm Entries N ∆IQ g3 ’U GF TR P N N g-G g3 F ’U PTR

B

N

A

15 10 5 0

Figure S4. Expression of AAV constructs in the dentate gyrus do not alter open field exploration or behavior in the elevated plus maze (Associated with Figure 4). (A-E). Behavioral validation of animals injected with eGFP-G3’UTR (n = 4) or eGFP- Ng3’UTR (n = 4). (F-J). Behavioral validation of animals injected with NgΔIQ-eGFP-Ng3’UTR (n = 3) or Ng-eGFP-Ng3’UTR (n = 4). Representative images (A,F), total distance traveled during 1h of exploration of open field (B,G);; percentage of time spent in center quadrant of the open field (C,H), number of open arm entries in 5 minutes in elevated plus maze (D,I), and percentage of time spent in the open arm of the elevated plus maze (E,J). Mean ± S.E.M is represented in solid black lines in each data column. Student’s t-test. All comparisons were not significant.

14

A WT Fmr1 KO n.s. stim n.s. stim mono L-poly H-poly

B

C Calm1

120

80

% mRNA

80 60

**

40

WT

60

20

m o L- no po H ly -p ol m y L- ono p H oly -p ol m y o L- no po H ly -p ol m y L- ono p H oly -p ol y

im

s.

n.s.

Fmr1 KO

stim

Gap43

% mRNA

60 40

*

n.s.

40 20 0

m o L- no po H ly -p ol m y L- ono p H oly -p ol m y o L- no po H ly -p ol m y L- ono p H oly -p ol y

% mRNA

80

stim

Fmr1 KO

n.s.

60

120 100

n.s.

WT

E

D

WT

im

st

s. n.

im

st

n.

s.

20 0

n.s.

40

st

st

n.

s.

0

**

0

n.

20

im

% mRNA

100

*

Fmr1 KO

n.s.

stim WT

n.s. stim Fmr1 KO

Figure S5. Calm1 and Gap43 mRNAs showed different distribution pattern and activity-sensitivity in the synaptoneurosome, compared to the Nrgn mRNA (Associated with Figure 6). (A). The data representation scheme. (B-E). Quantification of the stacking percentage (B,D) and the separated percentage (C,F) of the Calm1 mRNA (B,C) and the Gap43 mRNA (D,E) in monosome (mono), light polyribosome (L-poly), heavy-polyribosome (H-poly) fractions from non-stimulated (NS) and NMDA and glutamate (50 uM and 10 uM, NMDA+Glu) stimulated synaptoneurosome preps from wild-type (WT) and Fmr1 knockout (Fmr1 KO) animals, n = 3 in each group. Two-way ANOVA, (B), F(5,24) = 6.99, p = 0.0004;; (D), F(5,24) = 1.84, p = 0.14;; followed by Fisher’s LSD test for multiple comparison;; *, p < 0.05, **, p < 0.01.

15

B

FMRP Levels (% shLuc)

150

****

100

s N hLu g- c eG sh FP N Fm geG r1 FP

A

FMRP Tub

50

Ng-eGFP

r1 Fm

Ng

sh

sh

Lu

c

0

Figure S6. Kockdown of FMRP and knockdown of FMRP with Ng-eGFP overexpression (Associated with Figure 7). (A). Quantification of FMRP (normalized to Tub) in cortical neuron lysates from shLuc and shFmr1 infected neurons, n = 8 from 3 independent cultures. Mean ± S.E.M is represented in the bar graph. Student’s t-test **** p< 0.0001. (B). Representative western blot images of FMRP, Tub, Ng-eGFP, and Ng in cortical neuron lysates from shLuc-Ng-eGFP and shFmr1-Ng-eGFP infected neurons.

16

Table S1: Candidate genes selected for qRT-PCR with selection criteria and primer references (associated with Figure 2) Primer sequences for qRT-PCR, reference for primer sources and selection criteria for the candidate genes are listed. All candidate genes shown meet at least one of the following criteria: 1) Activity-dependent: activity-dependent or experience-dependent protein synthesis has been shown;; 2) Dendritic localization: Potential dendritic localization of the mRNA or direct evidence for dendritic localization has been shown;; 3) FMRP target: functionally validated as an FMRP target by at least two studies, and 4) Gene homology, whether the genes was homologous with another gene that met one or more of the three aforementioned criteria.

17

NCBI Accession

Gene Locus

Abbreviation

Tubb3

NM_023279

Tub

Actb

NM_007393

β-Actin

Nrgn

NM_022029

Ng

Calm1

NM_009790

CaM

Selection Criteria/Source

Reference /Source

Control

1

Actb F: TGTCACCAACTGGGACGATA Actb R: GGGGTGTTGAAGGTCTCAAA

Activity- dependent (2), Dendritic localization (2,3)

4

Nrgn F: CACCCTAACCTAACCTCAACC Nrgn R: AACCCAGTATGGTAACATGCA

Dendritic localization (5-8)

9

Control

10

Primer Tubb3 F: AGCCCTCTACGACATCTGCT Tubb3 R: ATTGAGCTGACCAGGGAATC

Calm1 1f: TTGCCGTCTATGACCACGTAAG Calm1 1r: CCTGCTTTTGCCATACACAGTG

Activity- dependent (11- 14), Dendritic localization (5,6,13,15), FMRP target (16-18) Camk2a homolog

Camk2a

NM_177407

CaMKIIα

Camk2a F: CCATCCTCACCACTATGCTG Camk2a R: ATCGATGAAAGTCCAGGCCC

Camk2b

NM_007595

CaMKIIβ

Camk2b F: GAAAGCAGATGGAGTCAAG Camk2b R: GTTGTGTTGGTGCTGTCG

Dlg1

NM_001252436

SAP-97

Dlg1 F: AGTGACGAAGTCGGAGTGATT Dlg1 R: GTCAGGGATCTCCCCTTTATCT

Dlg4 homolog

PrimerBank (21) ID 28502750a1

Dlg2

NM_001243047

PSD-93

Dlg2 F: GGGCTCTCTGCCCTAGAGTT Dlg2 R: AGCAGCTAAATCAGCTCTGGA

Dlg4 homolog

Primer blast

Dlg3

NM_001177778

SAP-102

Dlg3 F: ACATTCTGCACGTCATTAACGC Dlg3 R: ATGTCACTCCCTTCAGGTTCT

Dlg4 homolog


Activity- dependent (22), Dendritic localization (18), FMRP target (18,23-25)

Primer blast

Dendritic localization (26,27)

PrimerBank (21) ID 283549149c1

19

20

Dlg4

NM_001109752

PSD-95

Dlg4 F: GCCCTGAGCTTCCACTTTGG Dlg4 R: CCGCCGTTTGCTGGGAATGAA

Shank1

NM_001034115

Shank1

Shank1 F: TGCATCAGACGAAATGCCTAC Shank1 R: AACAGTCCATAGTTCAGCACG

Shank2

NM_001081370

Shank2

Shank2 F: GTTCCACATCCAAAGCCAAG Shank2 R: TGGGTGCACGTAATTCTCAG

Dendritic localization (27), Shank1 homolog

Primer blast

Shank3

NM_021423

Shank3

Shank3 F: TGGCAAGAGATCCATCAGG Shank3 R: GTTGGCCCCATAGAACAAAA

Dendritic localization (27), Shank1 homolog

Primer blast

Gria1

NM_001113325

GluA1

Gria2

NM_013540

GluA2

Grin1

NM_001177656

GluN1

Grin2a

NM_008170

GluN2A

Grin2a F: GACGGTCTTGGGATCTTAAC Grin2a R: TGACCATGAATTGGTGCAGG

Grin2b

NM_008171

GluN2B

Grin2b F: CAAGAACATGGCCAACCTGT Grin2b R: GGTACACATTGCTGTCCTTC

Bdnf

NM_001316310

BDNF

Activity- dependent (28- 30), Dendritic localization (18) Dendritic Gria2 F: 5' AAAGAATACCCTGGAGCACAC localization Gria2 R: 5' CCAAACAATCTCCTGCATTTCC (18,30,31), Gria1 homolog Gria1 F: GAGGTCCCGTAAACCTAGCG Gria1 R: GCTCAGAGCACTGGTCTTGT

Grin1 F: CTGCAACCCTCACTTTTGAG Grin1 R: TGCAAAAGCCAGCTGCATCT

Bdnf F: AGCTGAGCGTGTGTGACAGT Bdnf R: ACCCATGGGATTACACTTGG

Primer blast

Primer blast

Dendritic localization (30)

32

Dendritic localization (30), Grin1 homolog Dendritic localization (30), Grin1 homolog Activity- dependent (33,34), Dendritic localization (35)

32 32

19

18

Eef1a1 F: GCATCCTACCACCAACTGGT Eef1a1 R: CGTGTGGCAATCCAATACAG

Activity- dependent (36), Dendritic localization (36)

Primer blast

Control


Eef1a1

NM_010106

eEF1A1

Syp

NM_009305

Syp

Syngap1

NM_001281491

SynGAP1

Syngap1 F: TCCTGATGCAGTACCAAGCC Syngap1 R: CGGGTTTGTTGGACCCAAGG

Map1b

NM_008634

MAP-1B

Map1b F: CATCCTGCAGTCTGGCTCT Map1b R: AGGAGCTCACCAATCTCTTGA

Map2

NM_001310634

MAP-2

Gabrb3

NM_008071

GABRβ3

Gabrb3 F: CGTGGGTGTCCTTCTGGAT Gabrb3 R: ATGGTGAGCACGGTGGTAAT

Activity- dependent (43)

Primer blast

Dicer1

NM_148948

Dicer1

Dicer1 F: AGATGGAGGCGGAGTTCAG Dicer1 R: CAATGAGCAGGTTGGTCTCA

Activity- dependent (44)

Primer blast

Plat

NM_008872

tPA

Plat F: CTGAGGTCACAGTCCAAGCA Plat R: ACAGATGCTGTGAGGTGCAG

Ctnnb1

NM_007614

β-Cat

Ctnnb F: GTTGTACTGCTGGGACTC Ctnnb R: CAGTGTCGTGATGGCGTAGAACAG

Gapdh

NM_008084

GAPDH

Egr1

NM_007913

Egr-1

Fos

NM_010234

c-fos

Npas4

NM_153553

Npas4

Arc

NM_018790

Arc

Homer1

NM_011982

Homer1a

Rn18s

NR_046233

r18s

Syp F: CAGTTCCGGGTGGTCAAGG Syp R: ACTCTCCGTCTTGTTGGCAC

Map2 F: GCCAGCCTCGGAACAAACA Map2 R: GCTCAGCGAATGAGGAAGGA

Gapdh F: GCATCCTGCACCACCAACTG Gapdh R: ACGCCACAGCTTTCCAGAGG

Significant PSD- 95 interaction (37) Activity- dependent (17), Dendritic localization (16, 38,39), FMRP target (16, 40) Activity- dependent (41), Dendritic localization (42)

Activity- dependent (45- 46) Activity- dependent (48), FMRP target (49,50)

Primer blast

Primer blast

21

47

Primer blast

Control

19

IEG (51-54)

55

cfos F: ATGGGCTCTCCTGTCAACACAC cfos R: ATGGCTGTCACCGTGGGGATAAAG

IEG (56)

Primer blast

Npas4 F: GCTATACTCAGAAGGTCCAGAAGGC Npas4 R: TCAGAGAATGAGGGTAGCACAGC

IEG (65)

(57)

Arc F: CCCTGCAGCCCAAGTTCAAG Arc R: GAAGGCTCAGCTGCCTGCTC

IEG (58,59), Dendritic localization (6,60), FMRP target (61,62)

(19)

IEG (63)

Primer blast

rRNA marker

(4)

Egr-1 F: TATGAGCACCTGACCACAGAGTCC Egr-1 R: CGAGTCGTTTGGCTGGGATAAC

Homer1 F: CAAACACTGTTTATGGACTG Homer1 R: TGCTGAATTGAATGTGTACC r18s F: ACGGACCAGAGCGAAAGCAT r18s R: TGTCAATCCTGTCCGTGTCC

19

Table S2. Commercially Available Research Resource. Antibody dilutions for western blot (WB) and immunohistochemistry (IMH) were shown. Vendor name, catalog number, and available RRID info were listed. Antibodies Anti-Neurogranin WB: 1:3000 IHC: 1:500 (Fig 2G) Anti-Neurogranin IHC 1:200 (Fig 3I) Anti-Gapdh WB: 1:5000 Anti-Calmodulin WB: 1:3000 IHC: 1:500 Anti-Tubulin WB: 1:5000 Anti-hnRNP U WB: 1:1000 Anti-Synaptophysin IHC 1:1000 Anti-Beta Actin WB: 1:5000 Anti-FMRP WB: 1:1000 Anti-FXR2 WB: 1:1000 Anti-PUF60 WB 1:2000 Anti CaMKII WB: 1:5000 Anti-PSD-95 WB: 1:5,000 Anti-Puromycin IHC: 1:500 Goat anti-Mouse 680LT WB 1:10,000 Goat anti-Rabbit 800CW WB1:10,000 Alexa 488 Goat anti-Rabbit IHC: 1:1,000 Alexa 546 Goat anti-Mouse IHC: 1:1,000 Chemicals Anisomycin Cycloheximide Actinomycin ßD Bicuculline Methobromide Biotin-16-UTP Puromycin dihydrochloride Assays and Kits Doulink® In Situ Detection Kit Duolink® In situ anti-rabbit Plus

Millipore Cat#07-425;; RRID:AB_310605 Millipore Cat# AB5620 RRID:AB_91937 Millipore Cat# MAB374 RRID:AB_2107445 Millipore Cat# 05-173 RRID:AB_309644 Sigma-Aldrich Cat# T6074 RRID:AB_477582 Millipore Cat# 05-1516 RRID:AB_10563506 Sigma-Aldrich Cat# S5768 RRID:AB_477523 Sigma-Aldrich Cat# A2228 RRID:AB_476697 Abcam Cat# ab27455 RRID:AB_732400 Abcam Cat# ab65122 RRID:AB_1140561 Abcam Cat# ab184538 Epitomics Cat#2716-1 RRID:AB_2049246 Thermo Fisher Scientific Cat# MA1-046 RRID:AB_2092361 Millipore Cat#MABE343 RRID:AB_2566826 Licor Cat# 925-68020 Licor Cat# 925-32211 Thermo-Fisher Scientific Cat# A-11034 Thermo-Fisher Scientific Cat# A-11003 Sigma Cat#A9789 Sigma Cat#C7698 Sigma Cat#A9415 Tocris Cat#0109 Sigma Cat#11388908910 Sigma Cat#P8833 Sigma Cat#DUO92008 Sigma Cat#DUO92002

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Duolink® In situ anti-mouse Minus Puromycin dihydrochloride Streptavadin MyOne T1 Dynabeads Ambion MegaScript® T7 Transcription Kit Ambion MegaScript® T3 Transcription Kit Fugene® 6 Cell Lines HEK-293T Experimental Models: Organisms/Strains C57BL/6Ncrl FMR1 Knockout Software and Algorithms Scaffold 4 Reader Prism ImageJ Imaris Venny 2.1 Ethovision VersaMax Freezeframe/Freezeview

Sigma Cat#DUO92004 Sigma Cat#P8833 Thermo-Fisher Scientific Cat#65601 Thermo -Fisher Scientific Cat#1334 Thermo -Fisher Scientific Cat#1338 Promega E2692 ATCC Cat# CRL-11268, RRID:CVCL_1926 Charles River Laboratories Gift from Mark Bear Laboratory Originally from Jackson Labs Strain 003025 Proteome Software, Portland, Oregon GraphPad Software Inc. La Jolla, CA. NIH ImageJ Bitplane, Oxford Instruments http://bioinfogp.cnb.csic.es/tools/venny/ Noldus Software, Leesburg, VA. AccuScan Instruments Columbus OH. Actimetrics, Wilmette IL.

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Dataset S1: Proteins identified from hippocampal lysate interacting with the 3’UTR constructs Only proteins identified with at least 3 unique peptides were included in the table (A). Proteins interacting with Beads alone (background) (B). Proteins identified interacting with G3’UTR, but not beads alone (C). Proteins identified interacting with Ng3’UTR, but not beads alone (D). Proteins identified interacting with Ng3’UTR 389-577, but not beads alone (E). Proteins identified interacting with Ng3’UTR, but not beads, G3’UTR, or Ng3’UTR 389-577. Proteins shown in bold were identified in two independent mass spec samples, in this and subsequent sheets. (F). Proteins identified interacting with Ng3’UTR and Ng3’UTR 389-577, but not beads or G3’UTR. (G). Proteins identified exclusively in Ng3’UTR 389-577. (H). Proteins identified exclusively in G3’UTR. (I). Proteins identified interacting with Ng3’UTR and GAPDH3’UTR, but not beads, or Ng3’UTR 389-577. (J). Proteins identified interacting with Ng3’UTR 389-577 and GAPDH3’UTR, but not beads, or Ng3’UTR. (K). Proteins identified interacting with Ng3’UTR, Ng3’UTR 389-577, and GAPDH3’UTR, but not beads.

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