(A) Schematic of the Fmr1-cTag conditional knock-in mouse. The final exon of the Fmr1 gene is flanked by loxP sites and a second copy of this exon with an additional AcGFP sequence at the end of the …
Endogenous and GFP-tagged FMRP protein were detected in brain sections from P29 Fmr1-cTagCamk2a-Cre mice by immunofluorescence using antibodies against FMRP and GFP. The FMRP antibody (shown in …
(A) Immunoprecipitation of ribosome associated RNA from CA1 neurons from WT and Fmr1 KO animals. Differential analysis of counts per gene from TRAP RNA compared to input RNA showing fold change and …
The amount of antibody required for the TRAP IP was determined by titration. 500 μl hippocampal lysate was incubated with varying amounts of anti-HA antibody from 2.5 to 12.5 μg, followed by pull …
(A) Enrichment and depletion of relevant cell type markers in the immunoprecipitated TRAP mRNA compared to the input mRNA was confirmed using Gene Set Enrichment Analysis (GSEA). Cell type specific …
(A) Illustration of the CLIP score metric. For each biological replicate of CLIP, the density of CLIP tags across the coding region (log2 CLIP RPKM) of each transcript was plotted against the …
(A) CLIP score was determined either by normalization to mean TRAP RPKM per transcript or by normalization to mean FACS RNA-Seq RPKM per transcript. CLIP scores calculated with both methods were …
(A) Venn Diagram comparing FMRP targets in CA1 neurons to those determined from whole brain FMRP CLIP and cerebellar granule cell CLIP. Targets were defined as CLIP score >1 for CA1 and granule …
(A) Overlap of MeCP2-repressed genes (Supplementary Table 3 from Gabel et al., 2015) and CA1 FMRP targets with CLIP score > 1. Of the 466 MeCP2-repressed genes, 376 are expressed in CA1 pyramidal …
(A) Selected results from gene ontology analysis of CA1 transcriptome ranked by FMRP binding (CLIP Score) or negative log fold change Fmr1 KO vs WT TRAP. (B) Selected results from gene set …
(A) Selected results from gene ontology analysis of up-regulated genes in Fmr1 KO vs WT TRAP showing up-regulation of genes involved in protein synthesis and metabolism, mitochondrial function and …
RNA levels of the FMRP targets Ppargc1a, Npas2 and Ncoa2 are decreased in hippocampal tissue from Fmr1 KO mice relative to WT confirming TRAP results. Data is from 8 WT mice and 9 Fmr1 KO …
Fmr1 KO mice and WT littermates were euthanized at different times across the circadian cycle. Quantitative PCR was used to determine RNA abundance from CA1 tissue for four circadian transcripts …
(A) Schematic of spatial object recognition task. All mice were tested once during the day (9am – 11am) and once during the night (9pm – 11pm). Half of the cohort were tested first during the day …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain (M. musculus),strain background (C57BL6/J) | B6.Cg-Tg(Camk2a-cre)T29-1Stl/J | Jackson Laboratory | RRID:IMSR_JAX:005359 | Referred to as Camk2a-Cre. |
Strain (M. musculus),strain background (C57BL6/J) | B6N.129-Rpl22tm1.1Psam/J | Jackson Laboratory | RRID:IMSR_JAX:011029 | Referred to as RiboTag. |
Strain (M. musculus),strain background (C57BL6/J) | B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J | Jackson Laboratory | RRID:IMSR_JAX:007914 | Referred to as TdTomato. |
Strain (M. musculus),strain background (C57BL6/J) | B6.129P2-Fmr1tm1Cgr/J | Gift from W.T. Greenough. | RRID:IMSR_JAX:003025 | Referred to as Fmr1 KO. |
Strain (M. musculus),strain background (C57BL6/J) | Neurod1-cre (RZ24-CRE) | Gift from M.E. Hatten. | ||
Strain (M. musculus),strain background (C57BL6/J) | Fmr1-cTag | Van Driesche et al., 2019 | ||
Antibody | NeuN, guinea pig polyclonal | Millipore | Millipore Cat# ABN90P, RRID:AB_2341095 | For IF (1:2000) |
Antibody | GFP polyclonal antibody, rabbit | Invitrogen | Molecular Probes Cat# A-11122, RRID:AB_221569 | For IF (1:2000) |
Antibody | Anti-FMRP antibody, rabbit polyclonal | Abcam | Abcam Cat# ab17722, RRID:AB_2278530 | For IF (1:200) |
Antibody | Anti-HA tag, rabbit polyclonal | Abcam | Abcam Cat# ab9110, RRID:AB_307019 | For IP (20–80 µg/ml depending on Cre driver) |
Antibody | anti-GFP antibodies HtzGFP19C8 and HtzGFP19F7, mouse monoclonal | PMID: 19013281 | Heintz Lab; Rockefeller University Cat# Htz-GFP-19C8, RRID:AB_2716737 Heintz Lab; Rockefeller University Cat# Htz-GFP-19F7, RRID:AB_2716736 | For IP (25 µg each antibody for 1.2 ml lysate prepared from 8 to 10 animals) |
Antibody | Anti-BrdU, mouse monoclonal [IIB5] | Abcam | Abcam Cat# ab8955, RRID:AB_306886 | For IP (5 µg per pooled RT reaction) |
Antibody | anti-p0071, guinea pig polyclonal | Progen | Progen Cat# GP71 | p0071 is also known as Pkp4. For WB (1:1000) |
Antibody | Anti-ASCIZ, rabbit polyclonal | Millipore | Millipore Cat# AB3271, RRID:AB_11215293 | ASCIZ is also known as Atmin. For WB (1:5000) |
Antibody | p190-A RhoGAP, rabbit polyclonal | Cell Signaling | Cell Signaling Technology Cat# 2513, RRID:AB_2232820 | p190-A RhoGAP is also known as Arhgap35. For WB (1:1000) |
Antibody | Anti-HA tag, goat polyclonal | Abcam | Abcam Cat# ab9134, RRID:AB_307035 or Abcam Cat# ab215069, RRID:AB_2811264 | For WB (1:10,000) |
Antibody | Anti-Ribosomal P, human polyclonal | US Biological | US Biological Cat# R2031-25A, RRID:AB_2146244 | For WB (1:10,000) |
Software, algorithm | EthoVision tracking software | Noldus Information Technology | RRID:SCR_000441 | |
Software, algorithm | Behavioral Observation Research Interactive Software (BORIS) | doi: 10.1111/2041-210X.12584 | ||
Software, algorithm | FreezeFramethree software | Coulbourn Instruments | RRID:SCR_014429 | |
Software, algorithm | STAR | PMID: 23104886 | RRID:SCR_015899 | |
Software, algorithm | featureCounts | PMID: 24227677 | RRID:SCR_012919 | |
Software, algorithm | DESeq2, Bioconductor | PMID: 25516281 | RRID:SCR_015687 | |
Software, algorithm | CLIP Tool Kit (CTK) | PMID: 27797762 | ||
Software, algorithm | RSeQC | PMID: 22743226 | RRID:SCR_005275 | |
Software, algorithm | GenomicRanges, Bioconductor | PMID: 23950696 | RRID:SCR_000025 | |
Software, algorithm | Limma, Bioconductor | PMID: 25605792 | RRID:SCR_010943 | |
Software, algorithm | GOrilla | PMID: 19192299 | RRID:SCR_006848 | |
Software, algorithm | GSEA | PMID: 16199517 | RRID:SCR_003199 | |
Commercial assay or kit | Quant-iT RiboGreen RNA Assay Kit | ThermoFisher Scientific | Cat# R11490 | |
Commercial assay or kit | High Pure RNA isolation kit | Roche | Cat# 11828665001 | |
Commercial assay or kit | Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) | Illumina | Cat# MRZH11124 | |
Commercial assay or kit | Dynabeads mRNA Purification Kit | ThermoFisher Scientific | Cat# 61006 | |
Commercial assay or kit | TruSeq RNA library prep kit | Illumina | Cat# RS-122–2001 | |
Commercial assay or kit | iScript cDNA Synthesis Kit | Bio-Rad | Cat# 1708891 | |
Commercial assay or kit | FastStart SYBR Green Master | Roche | Cat# 04673484001 |
Summary of CA1 FMRP cTag CLIP read alignment.
Summary of read alignment for each replicate of FMRP cTag CA1 CLIP including Fmr1-cTagCamk2a-Cre and Fmr1-cTag negative control CLIP. The total number of reads obtained after demultiplexing and initial collapse of exact PCR duplicates is given (‘input reads for alignment’) as well as the number of these reads which uniquely mapped to the genome (‘Uniquely mapped to genome’) and transcriptome (‘Mapped within transcriptome and collapsed for PCR duplicates’). Finally, the number of reads which mapped to the positive strand of the CA1 transcriptome (as defined by CA1 TRAP rpkm > 1) is shown (‘Mapped within CA1 TRAP defined transcriptome for CLIP score determination’).
Summary of CA1 FMRP cTag CLIP and granule cell.
FMRP cTag CLIP data including counts per transcript, CLIP scores, FMRP target classifications and CA1 or granule cell-enriched binding. CA1 CLIP. CLIP tags that mapped within the coding region of each transcript were counted for each CLIP replicate and the final read count calculated by subtracting the number of CLIP reads in the Cre negative control from the number of CLIP reads from the Camk2a-Cre sample (CDS counts). These counts were converted to RPKM based on the length of the coding region. After normalization to abundance using TRAP RPKM per transcript as a proxy, the CLIP score for the transcript was calculated. CA1 Targets. List of transcripts classified as stringent, high binding or low-binding FMRP Targets in CA1 neurons. Stringent targets have a CLIP score > 2 in all three replicates, high binding targets have a mean CLIP score > 1 and low binding targets have a mean CLIP score between 0 and 1. Granule Cell CLIP. FMRP cTag CLIP was performed from cerebellar granule cells using Neurod1-Cre. Table of transcript information and CLIP scores is reproduced from Van Driesche et al. (2019). Granule Cell Targets. List of transcripts classified as stringent, high binding or low-binding FMRP Targets in cerebellar granule cells. Stringent targets have a CLIP score > 2 in all three replicates, high binding targets have a mean CLIP score > 1 and low binding targets have a mean CLIP score between 0 and 1. Enriched CA1. Transcripts with significantly higher binding in CA1 compared to cerebellar granule cells. Differential CLIP scores were determined using Limma. Log2 fold change and p-values are reported. Enriched Granule Cell. Transcripts with significantly higher binding in cerebellar granule cells compared to CA1 neurons. Differential CLIP scores were determined using Limma. Log2 fold change and p-values are reported.
Differential expression analysis of Fmr1 KO vs WT TRAP from CA1 neurons and cerebellar granule cells.
Results from Fmr1 KO vs WT TRAP. Raw counts per gene and statistics from DESeq2 analysis of differential expression between WT and KO are shown. Sawicka CA1 Fmr1 KO TRAP DESeq2. CA1 TRAP data from RiboTagCamk2a-Cre P28-P31 mice presented in this study. Ceolin CA1 Fmr1 KO TRAP DESeq2. CA1 TRAP data from RiboTagWfs1-CreERT22–6 month old mice published by Ceolin et al. (2017). Downloaded from GSE94559. Granule Fmr1 KO TRAP DESeq2. Cerebellar granule cell TRAP data from RiboTagNeurod1-Cre6–8 week mice presented in this study.
RT primers for CLIP Sequences for reverse transcription primers each containing a six nucleotide barcode index highlighted in red to allow pooling and multiplexing of samples for BrdU immunoprecipitation, PCR and sequencing.
Alternative CLIP normalization methods.
CLIP normalized to FACS RNA-Seq. CLIP scores determined from linear model using FACS RNA-Seq transcript RPKM. This table is equivalent to Supplementary file 2 CA1 CLIP but using RPKM per transcript from RNA-Seq of sorted CA1 pyramidal cells to normalize for abundance instead of TRAP. Counts and negative binomial. FMRP targets determined using a count-based method in which the FMRP CLIP read counts driven by mRNA abundance are modelled as following a negative binomial distribution. Reads from CLIP and TRAP replicates that mapped within the coding region of each transcript were counted. For CLIP the final read count was calculated by subtracting the number of CLIP reads in the Cre negative control (‘Neg CLIP CDS reads’) from the number of CLIP reads from the Camk2a-Cre sample (‘Camk2a CLIP CDS reads’). The mean TRAP counts normalized by library size across all three replicates are shown (‘mean normalized TRAP counts’). CLIP scores were derived from a linear regression model as the difference between the observed and fitted values. An estimated dispersion parameter for each CLIP count is given, along with individual p-values per replicate, combined p-value and FDR per transcript.