Regulation of Synaptic Structure and Function by FMRP- Associated MicroRNAs mir-125b and mir-132

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1 Neuron, Volume 65 Regulation of Synaptic Structure and Function by FMRP- Associated MicroRNAs mir-125b and mir-132 Dieter Edbauer, Joel R. Neilson, Kelly A. Foster, Chi-Fong Wang, Daniel P. Seeburg, Matthew N. Batterton, Tomoko Tada, Bridget M. Dolan, Phillip A. Sharp, and Morgan Sheng Legends for the Supplemental Figures Supplemental Figure S1 (related to Figures 2 and 4): Validation of mirna overexpression and knockdown in neurons. (A) β-actin promoter-driven mirna expression constructs suppress EGFP-based mirna sensors containing artificial mirna targets sites in their 3 UTR. Hippocampal neurons were cotransfected with mirna expression constructs and EGFP-based sensors at DIV4. Three days later, neurons were fixed, immunostained for EGFP and imaged by confocal microscopy. Co-expressed morange was used to pick neurons randomly for analysis (regardless of their EGFP-expression). mirna-expressing constructs downregulated only their specific mirna sensor but not unrelated sensors. One-way ANOVA with Dunnett s post test: p < 1. n = 15 to 55 neurons. Error bars denote SEM. (B) Design of mirna sponges. Diagram shows hybridization of mir-125b with the mir-125b sponge containing a central mismatch/bulge that prevents mrna cleavage by AGO2. In mirna sponge constructs, several such seed matches (yellow) were placed in the 3 UTR of mcherry. (C) mirna sponges specifically increase mirna-sensor expression in hippocampal neurons. mirna sponges were cotransfected with FF-luc-based mirna sensors (DIV4+3). Relative expression of FF-luc sensors was used to measure (residual) mirna activity in neurons. Note that a mir-125a-directed sponge also upregulated a

2 mir-125b sensor, presumably through cross-capture of mir-125b. Combining mir-125a and 125b sponges (125a+b) did not improve loss-of-function efficacy (as measured using the mir-125b sensor). One-way ANOVA with Dunnett s post test: * p < 5, p < 1. n = 12 to 24. Error bars denote SEM. Supplemental Figure S2 (related to Figure 2): Effects of overexpression of specific mirnas on dendrite branching. Sholl analysis of hippocampal neurons cotransfected with β-actin promoter-driven mirna expressing constructs and EGFP (DIV14+3). The number of dendrites crossing a series of concentric circles spaced 15 µm apart and centered upon the cell body of each neuron was manually counted. Gray corridor represents neurons transfected with empty vector (mean +/- SEM). mirna-expressing neurons were not significantly different from control using two-way ANOVA. n = 27 to 46 neurons each group. Error bars denote SEM. Supplemental Figure S3 (related to Figures 3 and 8): Validation of FMRP shrna constructs. (A) Validation of shrna constructs in HEK293 cells. Immunoblot showing shrnamediated specific knockdown of EGFP-FMRP in HEK293 cells cotransfected with the indicated psuper-based shrna constructs, and EGFP-FMRP and myc-psd-95 expressing constructs. (B) Cultured hippocampal neurons (DIV4+3) were cotransfected with psuper-based shrna constructs targeting zinc transporter 3 (ZnT3), EGFP or FMRP (two target sites) and mcherry. Neurons were immunostained for FMRP. Arrows mark cell body of transfected neurons. Scale bar represents 50 μm. (C) FMRP

3 immunostaining intensity was quantified in the soma of transfected relative to untransfected neurons in the same visual field. One-way ANOVA with Dunnett s post test compared to psuper: p < 1. n = 20 to 25 neurons. Error bars denote SEM. Supplemental Figure S4 (related to Figure 6): Validation of mirna expression constructs in HEK293 cells. FF-luc constructs containing perfect match mirna target sites in their 3 UTRs were cotransfected with synapsin promoter-driven mirna overexpression constructs and RRluc. Relative expression was determined by normalizing the ratio of FF-luc and RR-luc activity to the effect of each mirna on a control FF-luc sensor (and let-7c). Arrows indicate cognate sensor/mirna pairs. Note that let-7c, mir-22, mir-124 and mir-125b show no or low endogenous expression in HEK293 cells (Landgraf et al., 2007). One-way ANOVA with Dunnett s post test compared to let-7c overexpression: p < 1. n = 6 to 12. Error bars denote SEM. Supplemental Figure S5 (related to Figure 6): mirna target screening in HEK293 cells. Dual luciferase assays to screen 3 UTR reporter constructs from additional predicted mirna targets: Eph receptor A4 (EphA4), AMPA glutamate receptor 2 (GluR2), Arf guanine nucleotide exchange factor KIAA0522 (IQSEC2/BRAG1), N-cadherin, the protein phosphatase 1 and 2 catalytic subunits (PPP1CA and PPP2CA) and Profilin 2. For comparison data for NR2A is reproduced from Figure 5B. FF-luc reporter constructs containing the 3 UTR from the above genes were cotransfected with mirna overexpressing constructs (as indicated) and RR-luc. Relative expression was determined

4 by normalizing the ratio of FF-luc and RR-luc activity to the effect of each mirna on a control FF-luc sensor and, depending on the absence of predicted target sites, to let-7c (GluR2, N-cadherin, NR2A, PPP1CA, PPP2CA, Profilin 2) or mir-124 (IQSEC2, EphA4). Arrows indicate possible interaction between mirna and 3 UTR predicted by TargetScan or PicTar (Krek et al., 2005; Lewis et al., 2005). One-way ANOVA with Dunnett s post test: * p < 5, p < 1. n = 6 to 30. Error bars denote SEM. Supplemental Figure S6 (related to Figure 8): Developmental profile of mirnas and NR2 mrnas. Quantitative PCR analysis of NR2A, NR2B mrna (A) and mir-125b (B) from rat hippocampal neurons over the course of development in culture. Mature mir-125b levels were quantified by TaqMan assay. NR2A and NR2B mrna levels were quantified using SYBR green RT-qPCR. Expression levels are normalized to ζ (YWHAZ), which showed the most consistent expression pattern during development among the housekeeping genes tested (GAPDH, PGK1, YWHAZ). n = 3 7. Error bars denote SEM. Supplemental Figure S7 (related to Figure 8): Species-tagging of FMRP-associated mrnas. Single clone analysis to compare the ratio of rat and mouse cdna cloned from FMRPimmunoprecipitation and the input material (lysates of mixed WT rat and FMR1 KO mouse brain). (A) Colony PCR reaction of cdnas digested with rat-specific (left lane of each doublet) and mouse-specific (right lane of each doublet) restriction enzymes. NR1 was digested with BanII (for rat) and PflFI (for mouse); NR2A was digested with MboI (for rat) and BsrBI (for mouse). Clones were marked with R (rat cdna), M (mouse

5 cdna), x (cloning or colony PCR failed). Note that the size of the restriction fragments varies slightly depending on the orientation of the cdna insert in the cloning vector. (B) Clone counts for the indicated genes used for statistical analysis in Figure 8B. Supplemental Figure S8 (related to Figure 8): NMDA receptor subunit expression in FMR1 KO mice. Immunoblot analysis of hippocampus from FMR1 KO and wild-type mice for the indicated proteins at 7 days (A) or 14 days (B) of age. Immunoblots of homogenates were quantified by densitometry and normalized to total proteins levels (assayed with SYPRO Ruby) and wild-type mice. No significant changes were detected. (A) n = 5 mice each. (B) n = 14 wild-type and 12 KO mice. For each mouse the FMR1 genotype was confirmed by FMRP immunoblot. Error bars denote SEM.

6 A relative expression B mir-22 sensor 1.4 vector 9 22 mirna expression construct mcherry Ω relative expression Ω mir-125b sensor Ω vector b mirna expression construct CCA mir-125b 3 -AGUGUUCAAUC GAGUCCCU-5 sponge UCACAAGUUAG CUCAGGGA...-3 AA mirna Ω Ω_ C mir-124 sensor mir-125b sensor relative expression mirna sponge relative expression * a 125b125a+b mirna sponge Edbauer et al, Supplemental Figure S1

7 # of crossings distance from soma [µm] vector let-7c mir-22 mir-124 mir-132 mir-125b mir-143 Edbauer et al, Supplemental Figure S2

8 A myc-psd-95 B psuper ZnT3 FMRP #1 FMRP #2 EGFP C FMRP #2 EGFP Luc shrna EGFP-FMRP mcherry FMRP psuper ZnT3 FMRP #1 AGO1 psuper ZnT3 FMRP #1 FMRP #2 EGFP FMRP immunofluorescence Edbauer et al, Supplemental Figure S3

9 relative expression 1.4 control sensor mir-22 sensor mir-124 sensor mir-125b sensor let-7c mir-22 mir-124 mir-125b mirna overexpression Edbauer et al, Supplemental Figure S4

10 PPP2CA Profilin 2 NR2A PPP1CA 1.4 * relative expression EphA4 GluR2 let-7c mir-22 mir-124 mir-125b IQSEC2 N-cadherin Edbauer et al, Supplemental Figure S5

11 A 5 NR2A NR2B B relative expression days in vitro relative expression mir-125b days in vitro Edbauer et al, Supplemental Figure S6

12 A NR1: not significant R M M M M M M x M R M M x NR2A: p<01 R x R R R R M R R M R B input FMRP-IP M M M M M M M M x M M M R R R R R M R R M R x x mrna mouse rat mouse rat input FMRP-IP M M M M M x M M M x M R M M x x M M M x M M M M x M x M R M M R M M M M M M M M M R R M M M M M x M M M M M M M M M R R M M R M M M M M M M R M R R R R R R R R M R R M M R R R R R R M R x R R x M M x M M M M M R M R R M M M M M M M M M M M M M M M M x M M x R R R R R M M M x x M M M R x GAPDH MAP1B NR NR2A NR2B p Note: In the original publication, as a result of an error in the making of Figure S7, one of the gel panels was mistakenly duplicated and the correct panel omitted. The corrected version of the figure is shown here. The correction does not affect in any way the statistical analysis or conclusions of that experiment or the rest of the paper. The authors apologize for any confusion caused by this error. See the Erratum published in Neuron 68(1). Edbauer et al, Supplemental Figure S7

13 A B homogenate levels/protein homogenate levels/protein 2.0 WT 1.8 KO NR1 NR1 NR2A NR2A NR2B NR2B p250gap p250gap EphA4 PSD-95 MAP1B CaMK2a GluR1 b3-tubulin WT KO Edbauer et al, Supplemental Figure S8

14 Supplemental Table S1: DNA oligonucleotides. let- 7c mir- 22 mir- 124 mir- 125b mir- 132 mir- 143 mirna expression (first generation) s: GATCGTCGACCACTGGAAGCTGTGTGCATCCGGGTTGAGGTAGTAGGTTGTATGGTTTAGAGTTACACCCTGGG a: GATCGCGGCCGCCGATGGCTCAAGTGTGCTCCAAGGAAAGCTAGAAGGTTGTACAGTTAACTCCCAGGGTGTAACTCTAAACC s: GATCGTCGACCCACACGCTCACCTGGCTGAGCCGCAGTAGTTCTTCAGTGGCAAGCTTTATGTCCTGACCCAGCTAAAGC a: GATCGCGGCCGCCCTCCTCGAAGCCAGTGGCAGAGGGCAACAGTTCTTCAACTGGCAGCTTTAGCTGGGTCAGGACATAAAGC s: GATCGTCGACCTCAGGAGAAAGGCCTCTCTCTCCGTGTTCACAGCGGACCTTGATTTAAATGTCCATACAATTAAGGC a: GATCGCGGCCGCAGGTGCTCAGACAGCCCCATTCTTGGCATTCACCGCGTGCCTTAATTGTATGGACATTTAAATCAAG s: GATCGTCGACAGAATTGTGTTGCGCTCCCCTCAGTCCCTGAGACCCTAACTTGTGATGTTTACCGTTTAAATCCACG a: GATCGCGGCCGCCGATGCAAAGGCACGACTCGCAGCTCCCAAGAGCCTAACCCGTGGATTTAAACGGTAAACATCACAAG s: GATCGTCGACCCTGAAAGCCCCGCCCCCGCGTCTCCAGGGCAACCGTGGCTTTCGATTGTTACTGTGGGAACCGGAGGTAAC a: GATCGCGGCCGCGAGTGGTGGGGAGCGTGGGCGTGCTGCGGGGCGACCATGGCTGTAGACTGTTACCTCCGGTTCCCACAG s: GATCGTCGACTGGCCTGAGCGCGGAGCGCCTGTCTCCCAGCCTGAGGTGCAGTGCTGCATCTCTGGTCAGTTGGGAGTCTGAG a: GATCGCGGCCGCCCCGGCGACGGCTGCAGAACATCTTCTCCCTTCCTGAGCTACAGTGCTTCATCTCAGACTCCCAACTGACCAGAG mirna expression (second generation) let-7c s: GATGCTAGCCAGGTTAGGGTAGTCCTGTAAGCTAC a: CATGGCGCGCCAGGTCTGGGATGATCAGGTAAATGC mir-22 s: GATGCTAGCGAACCTGTGCCTCCCACACGCTC a: CATGGCGCGCCTTCCAGATGATAGGCAAAGAAGCTGC mir-124 s: GATGCTAGCCAACACTGCCAGCTTAGCGCGGAGCC a: CATGGCGCGCCGCATTGTTCGCCGGATTTGTCCGC mir-125b1 s: GATGCTAGCTTTACTCCTGTGTCCGCAACCGAG a: CATGGCGCGCCTCCTTCCTTCCTTAAAAGACAAAAAGTTCC mir-125b2 s: GATGCTAGCGCATGTCTGCTTTGTTTCCATAGCCC a: CATGGCGCGCCGAGGAAACACAAGCACTATTTCAGTACC mir-143 s: GATGCTAGCGGGTAGGGTCCATCTCAAGAAAGC a: CATGGCGCGCCAGCCCTGGTACAGCCTGAGTCAAGG Mature mir-125b of identical sequence is expressed from two genomic loci mir-125b1 and mir-125b2. We found that the mir-125b1-derived vector allows stronger lentiviral mir-125b expression (Figure 7A), while the mir-125b2 derivative allows stronger expression in HEK293 cells (Figure 6B and Supplemental Figures S4 and S5). mirna sponge cloning (p denotes 5 phosphorylation, bold indicates mismatch with the corresponding mirna) 5 -linker s: AGTCACTAGTCTTGATGCGGCCGCTc a: ccgggagcggccgcatcaagactagtgact 3 -linker s: p-ccggcgtcgactcggcagctctagacgc a: GCGTCTAGAGCTGCCGAGTCGACg let-7c s: p-ccggcaaccatacaacagctacctcac a: ccgggtgaggtagctgttgtatggttg mir-22 s: p-ccggcacagttcttcagaggcagcttc a: ccgggaagctgcctctgaagaactgtg mir-124 s: p-ccggctggcattcacaagtgccttaac a: ccgggttaaggcacttgtgaatgccag mir-125a s: p-ccggccacaggttaaagaactcagggac a: ccgggtccctgagttctttaacctgtgg mir-125b s: p-ccggctcacaagttagaactcagggac a: ccgggtccctgagttctaacttgtgag

15 mir-132 mir-143 s: p-ccggccgaccatggctaggactgttaac a: ccgggttaacagtcctagccatggtcgg s: p-ccggctgagctacagtagtcatctcac a: ccgggtgagatgactactgtagctcag Primer pairs for qpcr (bispecifc for mouse and rat) p250gap s: TGTGGGGCAGGTACAAGAAGCACC a: TGAAAAAGGAACGCCAGCTGCCAAC NR1 s: TCTGGCCAGGAGGAGAGACAGAG a: TGTCATTAGGCCCCGTACAGATCACC NR2A s: GGGCTGCTCTTCTCCATCAGC a: CCCTTGTCTGAAACCATGTCCAC NR2B s: GGAAGCTCTCTGGCTCACTGGC a: TCATCACGGATTGGCGCTCCTC YWHAZ s: TGAGCAGAAGACGGAAGGTGCTG a: TCTGATGGGGTGTGTCGGCTGC MAP1B s: TCCGATCGTGGGACACAAACCTG a: AGCACCAGCAGTTTATGGCGGG PGK1 s: GAAGGGGAAGCGGGTCGTGATG a: GCAGCAACTGGCTCTAAGGAGTACTTG GAPDH s: CCGCATCTTCTTGTGCAGTGCC a: AGACTCCACGACATACTCAGCACC 3 UTR cloning for FF-luc reporter constructs N-cad- s: ATCTCTAGAACGGCAGGACGGACTTGGCTTTTGG herin a: CATGAATTCCAAAATTAGCTTTTTATTCAGAACGCTGGG EphA4 s: ATGGGCGCGCCAGTACCGAACAGACTCAAAACTCTCG a: TTGGAATTCGTATGAAACAAGAGATGTCCGGCTAACTTC GluR2 s: GATGCTAGCTTAGGGGATGACCTTGAGTGATGTCATGAGG a: TAAGAATTCTGGCTGAAAGAAAAGCCTTCAAGATATCGG IQSEC2 s: GCGTCTAGACAGTCCACGCCCTCACCAGAGGATGC a: CATGAATTCCTGTCTGGACTTTGATCAGGATAAGAAC NR1 s: GATGCTAGCGTCATAGGGAGAGCTGAGACGCCC a: ATGGAATTCCATAGGCCCCCACTGTAGCTTGG NR2A s: ATGTCTAGACGAGTGTACAAGAAAATGCCTAGTATCG a: CATGAATTCATATGCACAGGCTCAGTGGGCC NR2B s: GATGCTAGCTAGTATTGAGTCTGATGTCTGAGTGAGGG a: GATGAATTCAGTGAAAGCCTCTTCTCTCTTATG Profilin s: GATTCTAGAGCTAGGCAGACTGTTAAGTATTAGGGG 2 a: ATTGAATTCATCTGTCTGTAAACAAGGTGTTTAATAGTTATGG PPP1CA s: GCGTCTAGACCTCCATGTGCTGCCCTCTGCC a: CATGAATTCAAGAGACCAGATGGGTTGCCCCAGG PPP2CA s: GATTCTAGACACATGTCACTCGTCGTACCCC a: CCTGGAATTCTACAAATTCTGAAATTGATC