. 2020 Mar 31;30(13):4459-4472.e6.
FMRP Control of Ribosome Translocation Promotes Chromatin Modifications and Alternative Splicing of Neuronal Genes Linked to Autism
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FMRP Control of Ribosome Translocation Promotes Chromatin Modifications and Alternative Splicing of Neuronal Genes Linked to Autism
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Silencing of FMR1 and loss of its gene product, FMRP, results in fragile X syndrome (FXS). FMRP binds brain mRNAs and inhibits polypeptide elongation. Using ribosome profiling of the hippocampus, we find that ribosome footprint levels in Fmr1-deficient tissue mostly reflect changes in RNA abundance. Profiling over a time course of ribosome runoff in wild-type tissue reveals a wide range of ribosome translocation rates; on many mRNAs, the ribosomes are stalled. Sucrose gradient ultracentrifugation of hippocampal slices after ribosome runoff reveals that FMRP co-sediments with stalled ribosomes, and its loss results in decline of ribosome stalling on specific mRNAs. One such mRNA encodes SETD2, a lysine methyltransferase that catalyzes H3K36me3. Chromatin immunoprecipitation sequencing (ChIP-seq) demonstrates that loss of FMRP alters the deployment of this histone mark. H3K36me3 is associated with alternative pre-RNA processing, which we find occurs in an FMRP-dependent manner on transcripts linked to neural function and autism spectrum disorders.
Fragile X Syndrome; alternative splicing; autism; chromatin modifications; ribosome stalling.
Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.
Conflict of interest statement
Declaration of Interests The authors declare no competing interests.
Figure 1.. Ribosome Profiling Reveals Diverse Changes of Gene Expression in Fmr1 KO Hippocampus
(A) Schematic diagram of the hippocampal slice preparation. To reduce spontaneous electrical activity, region CA3 was excised. 8–10 slices from six to eight mice/genotype were pooled per biological replicate (wild-type [WT], n = 4 and
Fmr1 KO, n = 3). (B) Schematic diagram of the experimental procedures for ribosome profiling. (C) Scatterplot of expression changes of mRNA levels and ribosome-protected fragments (RPFs). Dysregulated mRNAs in the absence of FMRP are classified into four regulatory groups. 14,459 genes past filtering are used for the scatterplot. Nominal p < 0.01, FDR = 0.097 by permutation test. FC, fold change; TE, translational efficiency. (D) Top three GO terms of Biological Process enriched in each regulatory group. The enrichment (gene ratio) is represented by the size of dots. The enrichment significance (adjusted p value) is colorcoded. (E) Top three GO terms of Cellular Component enriched in each regulatory group. See also Figure S1 and Table S1.
Figure 2.. Characterization of Genes with TE Changes in Fmr1 KO Hippocampus
(A) Heatmap of expression changes (log
2FC KO/WT) for the top 20 RNAs in the “TE up” group. (B) Heatmap of expression changes (log 2FC KO/WT) for the top 20 RNAs in the “TE down” group. (C) Read distributions on the Gfpt2 RNA of TE-up group. Normalized RPF reads (top) and mRNA reads (bottom) averaged across all replicates are plotted along the mRNA nucleotide positions with green and red triangles for annotated start and stop codons, respectively. For visualization purposes, the curves were smoothed within a 30-nt window. (D) Read distributions on the Lhfpl2 RNA of the TE-down group. (E) Boxplots visualize the medians of expression changes for FMRP CLIP targets. The lower and upper hinges correspond to the first and third quartiles. The whiskers extend from the hinges to the largest and smallest values no further than 1.5-fold of interquartile range. Outliers are not shown. Gene expression changes of CLIP genes were compared to those of all genes used for differential expressed genes (DEG) analysis (ns, not significant; ****p < 0.0001; Wilcoxon test). CLIP genes are the FMRP targets identified in (Darnell et al., 2011). All grouped data are presented as mean ± s.e.m. (F) Overlap of the TE-down and TE-up genes detected in this study (orange) with those of Das Sharma et al. (2019) (green) and the TRAP-seq data of Thomson et al. (2017) (blue). (G) Schematic models of ribosome density (TE) changes that reflect increased protein synthesis rates. See also Table S1.
Figure 3.. Runoff Ribosome Profiling of WT Mouse Brain Slices
(A) Schematic diagram of homoharringtonine (HHT) runoff ribosome profiling. WT mouse brain slices were treated with 20 mM HHT, an inhibitor of translation initiation, to allow ribosome runoff for 5, 10, 30, and 60 min (t) at 30°C. 10 slices from two or three mice were pooled per time point of HHT treatment. (B) Metagene plot of RPFs after HHT treatment. Reads are mapped transcripts (n = 1,401) with CDS longer than 3,000 nt and aligned at the annotated start and stop codons (gray vertical dash lines). The read densities at each nucleotide position are normalized to the average density of the last 500 nt of CDS and averaged using the P sites of RPFs. For visualization purposes, the curves were smoothed within a 90-nt window. Black horizontal dash line indicates the arbitrary 0.8 threshold to estimate the relative runoff distances (black vertical dash lines). (C) Linear regression between the HHT treatment time and ribosome run-off distances (from B) to estimate the global elongation rate (4.2 nt/s). (D) Cluster analysis of gene groups with distinct ribosome runoff patterns. The RPFs of each gene at each time point was normalized to time 0. The Euclidean distance matrix was then calculated, followed by hierarchical clustering using Ward’s agglomeration method (Ward, 1963). (E) Ribosome runoff patterns for each subcluster. The global pattern of each subcluster was summarized using the corresponding median and standard deviation in each time point. The number of RNAs in each subcluster is shown in parentheses. (F) Representative ribosome runoff profiles that reflect each subcluster. The runoff pattern for
Actin is similar to subclusters 4–6. (G) Ribosome footprints for Nrxn3 and Actin mRNAs during the runoff time period. (H) GO terms for subclusters 1 and 3. Gene ratio refers to the percentage of total differentially expressed genes in the given GO term. See also Figure S2 and Table S1.
Figure 4.. Substantial FMRP Remains Associated with Polysomes after HHT Treatment
(A–D) WT mouse hippocampal slices were treated with DMSO vehicle (A) or 20 mM HHT (B) for 30 min at 30°C. In parallel,
Fmr1 KO mouse hippocampal slices were also treated with DMSO vehicle (C) or 20 mM HHT (D) for 30 min at 30°C. Slices were homogenized and applied to 15%–45% (w/w) sucrose gradients, which were fractionated with continuous monitoring of A 260 after ultracentrifugation. Fractions were collected for immunoblotting with indicated antibodies to detect the association of FMRP with polysomes. 10 slices from two or three mice/genotype were pooled per biological replicate (WT, n = 2; Fmr1 KO, n = 2). (E) RNA sedimenting to heavy fractions containing more than seven ribosomes from WT slices treated with HHT was analyzed relative to input. 1,574 RNAs were reduced and 686 were elevated relative to input after HHT treatment. (F) Actin mRNA reads in the designated conditions (WT; V, vehicle, I, input; H, heavy fractions; KO, Fmr1 KO). (G) Map 1b mRNA reads in the designated conditions. (H) GO terms of RNAs depleted in heavy fractions relative to WT. (I) GO terms of RNAs enriched in heavy fractions relative to WT. See also Figure S3 and Table S1.
Figure 5.. FMRP Stalls Ribosomes on Specific mRNAs
(A) RNA sedimenting to medium polysomes containing four to six ribosomes after HHT treatment of hippocampal slices; 46 RNAs are downregulated and 1 is upregulated in
Fmr1 KO relative to WT. (B) Downregulated RNAs in HHT-treated Fmr1 KO slices primarily encode epigenetic and transcriptional regulators and proteins involved in neural function. (C) Example of Ankrd12 RNA, which has reduced reads in Fmr1 KO slices relative to WT after HHT (H) treatment. Input (I) reads are similar in both genotypes. M refers to medium fraction. (D) Boxplot showing the fold change of Fmr1 KO versus WT of all RNAs (white) compared to those identified in (A) and (B) (gray) with respect to steady-state RNA levels, RPFs, and TE (ns, not significant; **p < 0.01; ***p < 0.001; Wilcoxon test). All grouped data are presented as mean ± s.e.m. (E) Western blot analysis of SETD2 and lamin AC in hippocampus from four WT and five Fmr1 KO mice. When quantified and made relative to lamin AC, SETD2 was significantly increased in the KO (p = 0.0245, two-tailed t test). All grouped data are presented as mean ± s.e.m. See also Table S1 and Figure S5E.
Figure 6.. H3K36me3 Localization Is Altered in Fmr1 KO Hippocampus
(A) Experimental design for
in vivo ChIP-seq of H3K36me3 in hippocampus from adult WT (n = 4) and Fmr1 KO (n = 4) mice. (B) Pie chart representing the genomic annotation of the total H3K36me3 islands identified in the WT and Fmr1 KO ChIP-seq. (C) H3K36me3 ChIP-seq gene tracks for WT and Fmr1 KO hippocampal tissue. The two sequencing tracks from each biological replicate (n = 2 [pooled hippocampi from two mice/biological replicate]) of WT and Fmr1 KO were merged and overlaid. WT ChIP-seq tracks are in blue, and KO tracks are in green. The tracks for immunoprecipitation (IP) and input are displayed. The islands with significantly decreased (blue) or increased (green) tracks are shown below the RefSeq gene annotation (FDR < 0.0001; p < 0.01) as identified using the SICER package. Reep4 shows decreased H3K36me3 islands in Fmr1 KO, and Tprkb shows increased islands in Fmr1 KO. (D) Distribution of significantly increased or decreased H3K36me3 islands in intragenic and intergenic regions of the genome using negative binomial test performed using edgeR (Robinson et al., 2010) (fold change >2 and p <0.05). The total number of increased or decreased islands is indicated above the respective bars in the graph in Fmr1 KO versus WT ChIP-seq. (E) Venn diagram for significant overlap of Fmr1 KO misregulated H3K36me3 genes (increased islands in blue and decreased islands in green) with the ASD-linked genes from the SFARI database. A subset of genes showed both increased and decreased islands along the length of the gene body. The p value (hypergeometric test) is indicated next to the respective comparisons. (F) GO term enrichment of H3K36me3 differentially enriched genes in Fmr1 KO versus WT (p adjust value < 0.05). Gene ratio refers to the percentage of total differentially expressed genes in the given GO term. See also Figure S4 and Table S2.
Figure 7.. Global Analysis of FMRP-Mediated Alternative Splicing
(A) Summary table of total splicing events in
Fmr1 KO and WT based on RNA-seq from hippocampal slices. Splicing events detected by rMATS at a FDR <5% and a difference in the exon inclusion levels between the genotypes (deltaPSI) ≥5% are depicted. (B) Alternative splicing events validated using qRT-PCR are shown for several RNAs (hippocampus tissue, WT, n = 6; Fmr1 KO, n = 6). The illustration depicts an example of exon skipping (green box) in the Fmr1 KO. Primer positions are depicted with black bars. All grouped data are presented as mean ± s.e.m. (C) Alternative splicing events detected in Fmr1 KO and WT hippocampus. Inclusion events that were significantly (p < 0.05) increased (red), decreased (blue), or unchanged (gray) are indicated. The numbers of events in the up or down category are shown in red and blue, respectively. PSI, percent splice in/exon inclusion levels. (D) GO term enrichment for all alternative splicing events are shown (p adjust value < 0.05). The total number of genes identified in the RNA-seq from (Figure 1) was used as background. Gene ratio refers to the percentage of total differentially expressed genes in the given GO term. (E) Table depicting the overlap of the alternative splicing events (Alt. Spl.) in genes in this study with the SFARI autism spectrum disorder database, the Alt. Spl. genes identified in samples from autism patients (Parikshak et al., 2016), and genes with increased or decreased H3K36me3 islands from Figure 6. The intensity of the color represents the increasing number of overlapping genes between the gene sets. Asterisks indicate statistical significance (*p < 0.05; ***p < 0.001; ****p < 0.0001, hypergeometric test). (F) Violin plot for the H3K36me3 ChIP signal at ±50 nt of the 5 ′ (SS5) and 3 ′ (SS3) splice sites of the alternatively skipped exons in WT (white) and Fmr1 KO (red) hippocampus tissue (p < 0.05, K-S test for significance). See also Figures S5E and S5F. (G) Model for FMRP-mediated alterations in H3K36me3 marks on the chromatin and alternative splicing of transcripts in the hippocampus. See also Table S3.
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