Dilated cardiomyopathy-associated RNA-binding motif protein 20 regulates long pre-mRNAs in neurons
- Biozentrum, University of Basel, Switzerland
- Department of Experimental Cardiology, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Netherlands
Figures
Figure 1 with 2 supplements
Characterization of Rbm20 expression in the brain.
(A) Dot plot of the expression of a hand-curated list of RNA-binding proteins (RBPs) across different neuronal neocortical populations. RBPs were chosen based on the presence of an RNA recognition motif (RRM) in their sequence and on the ranking of their gini-index value (only the first 20 RBPs displaying the highest gini-index value are displayed [see Materials and methods]). RBPs’ expression was measured by Ribo-TRAP sequencing and expressed as RPKM values normalized over the mean expression across different neuronal populations. (B) Sagittal section of the mouse brain used for immunohistochemistry of RBM20 (gray). The somatosensory cortex and the olfactory bulb regions, where RBM20 is expressed, are highlighted. Scale bar: 1 mm. (C) Immunohistochemistry of RBM20 expression (green) in Parvalbumin-positive interneurons (red) in the neocortex. (D) Schematic illustration of the olfactory bulb circuitry and cell types (left). GL: glomerular layer, EPL: external plexiform layer, MCL: mitral cell layer, GCL: granule cell layer (left). RBM20 expression (green) is specific to the mitral cell layer and glomeruli layer of the olfactory bulb identified in the Slc17a6Cre:: Rpl22HA mouse line (HA staining in red) (middle and right). Scale bar: 100 µm. (E) Western blot probing RBM20 expression in olfactory bulb and cortex samples of wild-type (WT) and constitutive Rbm20KO mice (KO). The RBM20 band at ca. 150 kDa indicated with an arrow is selectively lost in KO tissue. GAPDH detection is used as loading control. For better visualization of the two proteins, the same tissue lysates were run on a 7.5% acrylamide gel (for RBM20 detection) and 4–20% acrylamide gradient gel (GAPDH). (F) Immunofluorescence of RBM20 (green) expression in the mitral cell layer (MCL) of P0 and P35 Tbx21Cre:: Rosa26LSL-tdTomato mice. Mitral cells and tufted neurons are labeled with the tdTomato reporter (red). Scale bar: 100 µm.
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Figure 1—source data 1
PDF file containing original western blots for Figure 1E, indicating the relevant bands and conditions.
- https://cdn.elifesciences.org/articles/104808/elife-104808-fig1-data1-v1.zip
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Figure 1—source data 2
Original files for western blot analysis displayed in Figure 1E.
- https://cdn.elifesciences.org/articles/104808/elife-104808-fig1-data2-v1.zip
Figure 1—figure supplement 1
Rbm20 mRNA expression in mouse neocortex.
(A) Dot plot of the expression of a hand-curated list of RNA-binding proteins (RBPs) across different neuronal neocortical populations. RBPs were chosen based on the presence of an RNA recognition motif (RRM) in their sequence and their expression in the neocortex. RBPs expression was measured by Ribo-TRAP sequencing and expressed in RPKM values normalized over the mean expression across different neuronal populations. (B) Fluorescent in situ hybridization (FISH) on P25 brain slices from mice with genetic marking of cell populations (PvalbCre::Rosa26LSL-tdTomato Cre-dependent tdTomato expression). Red: tdTtomato mRNA, blue: DAPI. Scale bar: 100 μm. (C) FISH for Rbm20 transcripts in tdTomato-marked cells for different cell classes (Cre-dependent tdTomato reporter crossed to the indicated Cre-recombinase expressing lines: Camk2, Pvalb, Sst, Vip) in P23–26 somatosensory cortex cells in layer 5. Green: Rbm20 mRNA, red: tdTtomato mRNA, blue: DAPI. Scale bar: 10 μm. (D) Quantification of Rbm20 mRNA expression as in C, expressed as mean number of fluorescent dots per cell from three animals per genotype. n=3; p-Value<0.01, one-way ANOVA. (E) Western blot analysis of RBM20 expression in cortex (CX), olfactory bulb (OB), and heart (HR) samples from WT and constitutive KO mice. An RBM20 antibody cross-reacting band with apparent mobility of ca. 150 kDa is lost in the KO samples (indicated with an arrow). Anti-GAPDH detection serves as a loading control.
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Figure 1—figure supplement 1—source data 1
PDF file containing original western blots for Figure 1—figure supplement 1E, indicating the relevant bands and conditions.
- https://cdn.elifesciences.org/articles/104808/elife-104808-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 1—figure supplement 1E.
- https://cdn.elifesciences.org/articles/104808/elife-104808-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
Rbm20 mRNA expression in mouse olfactory bulb.
(A) Schematic illustration of the olfactory bulb circuitry and cell types. GL: glomerular layer, EPL: external plexiform layer, MCL: mitral cell layer, GCL: granule cell layer, (left). Fluorescent in situ hybridization (FISH) on brain slices for Rbm20 (green), Tbr2 (gray), Slc17a6 (red) mRNAs (middle). The insets show the example of a cell expressing all three markers (right). Scale bar: 100 μm; scale bar insets: 10 μm. (B) Pie charts indicating the quantification of the percentage of cells of the MCL and GL expressing Slc17a6, Rbm20, and Tbr2 transcripts. Among the Rbm20+ cells, the percentage of neurons presenting co-localization with glutamatergic markers was calculated. (C) Quantification of the percentage of neurons of the mitral cell layer (MCL) and glomerular layer (GL) expressing high or low Rbm20 mRNA levels, co-localizing with Tbr2 and Slc17a6 markers. The absolute number of high and low Rbm20 expressing neurons identified in both the mitral cell layer and the glomeruli layer is reported in brackets. (D) FISH on brain slices of Rbm20 (green), Gad1 (gray), Slc17a6 (red) mRNAs. Scale bar: 100 µm. The arrow in the inset on the right of the panel shows an example of a cell expressing low Rbm20 levels and co-localizing with Gad1 but not Slc17a6 mRNAs. Scale bar inset: 10 μm.
Figure 2 with 1 supplement
Identification of RBM20 direct targets in the heart and olfactory bulb.
(A) Subnuclear localization of RBM20 (green) in heart cardiomyocytes (left) and mitral cells of the olfactory bulb (right) of wild-type (WT) mice at P35. Scale bar: 10 µm. (B) Schematic illustration of HA epitope tagging of endogenous RBM20 in mice. A cassette was inserted into the Rbm20 locus containing a strong synthetic 3’ splicing acceptor site (3’SA) introduced into sequences derived from Rbm20 exon 14 and in-frame fusion of a histidine-biotin-histidine-3xHA tag, followed by a polyadenylation signal (up). Schematic representation of the resulting RBM20HA protein where the last exon of the protein is fused to a histidine-biotin-histidine-3xHA tag (down). (C) Western blot showing the validation of RBM20 expression in the olfactory bulb and cortex tissues of WT and Rbm20HA tagged mice. GAPDH is used as a loading control. (D) Quantification of the percentage of peaks identified in the heart (red) and (E) olfactory bulb (blue) tissue in each genomic feature: introns, untranslated regions (3’UTR, 5’UTR), coding sequence (CDS), others (promoters, intergenic regions, noncoding regions). The absolute number of the peaks identified in each genomic feature is reported on top of the corresponding bar. (F) Bar plot showing the percentage of peaks identified in distal (>500 bp) or proximal (<500 bp) intronic regions in the heart (red) and olfactory bulb (blue). The absolute number of the peaks identified is reported on top of the corresponding bar.
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Figure 2—source data 1
PDF file containing original western blots for Figure 2C, indicating the relevant bands and conditions.
- https://cdn.elifesciences.org/articles/104808/elife-104808-fig2-data1-v1.zip
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Figure 2—source data 2
Original files for western blot analysis displayed in Figure 2C.
- https://cdn.elifesciences.org/articles/104808/elife-104808-fig2-data2-v1.zip
Figure 2—figure supplement 1
Generation of Rbm20 – HA-tagged mouse line.
(A) Schematic illustration of the ‘COIN allele’ strategy used for generation of Rbm20HA knock-in mice. The COIN allele module was inserted in the Rbm20 locus with the CRISPR-CAS9 system. Upon Cre-mediated recombination of the loxP sites, the COIN module is inverted and the presence of a strong synthetic 3’ splicing acceptor site (3’SA) results in the expression of the tagged Rbm20 isoform. We observed that the efficiency of Cre-mediated inversion of this allele was too low for conditional tagging in Cre-recombinase-expressing mouse lines. Thus, an allele with germline recombination was generated, resulting in constitutive tagging of RBM20 protein in all cells. (B) Immunohistochemistry of RBM20HA protein in mitral cells of wild-type (WT) and Rbm20HA mice. HA (gray), DAPI (blue). Scale bar: 10 μm. (C) Targeted proteomic analysis on heart samples of WT, Rbm20WT/HA heterozygous, and Rbm20HA/HA homozygous knock-in mice (n=4). Three proteotypic peptides were quantified. Plotted results represent means of all three peptides normalized to WT samples. Four biological replicates per genotype were used. The mean of the –log2 ratio (light/heavy) peptides was calculated and displayed. Note that an assessment of RBM20 expression level in the Rbm20HA mice by western blot was not possible as the C-terminal HA epitope reduced binding affinity of the anti-RBM20 antibody raised against the C-terminus of the protein.
Figure 3 with 1 supplement
Identification of RBM20 direct mRNA targets in the heart and olfactory bulb.
(A) Tracks illustrating RBM20 CLIP-seq signal for Ttn, CamkIId, Cacna1c, and Nav2. Read density obtained for heart samples (red traces) and olfactory bulb (green traces) with the corresponding input samples (overlaid traces in gray). CLIP peaks, considered statistically significant by irreproducible discovery rate (IDR) (score>540, equivalent to IDR<0.05), are marked by black arrowheads. RBM20-dependent alternative exons previously reported for cardiomyocytes are labeled in red. Note that in the olfactory bulb, RBM20-binding sites are identified on CamkIId and Cacna1c pre-mRNAs. However, these binding sites are distal (>500 bp) to the alternative exons. See Supplementary file 1 for coordinates. (B) Illustration of the gene ontology (GO) categories of RBM20 mRNA targets in the heart (IDR<0.05). Cellular component analysis with Bonferroni correction (p-value≤0.05). The number of genes found in each category is displayed on top of each bar. Minimal number of genes identified in each category: five genes. (C) Illustration of the GO categories of RBM20 mRNA targets in the olfactory bulb (IDR<0.05). Cellular component analysis with Bonferroni correction (p-value≤0.05). The number of genes found in each category is displayed on top of each bar. Minimal number of genes identified in each category: five genes.
Figure 3—figure supplement 1
Identification of RBM20-binding sites on transcript mRNAs.
(A) Anti-HA western blot for input (left) and immunoprecipitated HA-tagged RBM20 recovered from Rbm20HA/HA knock-in mice with precipitations performed after treatment with multiple RNaseA concentrations (25–100 units/ml) with (+) or without (-) UV-cross-linking of RNA-protein complexes. (B) Agarose gel of libraries resulting from processed anti-HA IP samples amplified with 22 PCR cycles (using Illumina D701 and D501 primers). A negative control sample (water input instead of precipitated RNA) is shown on the right. (C–F) Correlation analysis of normalized counts (reads per million [rpm]+1) of called CLIP peaks between seCLIP replicates of the heart (2 samples, panel A) and the olfactory bulb (3 samples, panels B, C, D). Gray shades represent density of the data points. Pearson coefficients are indicated above the corresponding plots. (G) Enrichment of the TCTT motif at cross-link-induced truncation sites (CITS) in both heart and olfactory bulb tissues. The enrichment is calculated from the frequency of the TCTT motif starting at each position of the inferred cross-linked site, normalized by frequency of the same motif in adjacent flanking regions. (H) Motif finding analysis performed with DREME on heart and olfactory bulb seCLIP datasets. The first five statistically significant motifs are reported, ranked based on the enrichment p-value (E-value; right of each panel). The E-value is defined as the p-value times the number of candidate motifs tested. The enrichment p-value is calculated using Fisher’s exact test for enrichment of the motif in the positive sequences, calculated after erasing sites that match previously found motifs. Note that the G-rich motif (rank 3) is commonly found to be nonspecifically recovered in seCLIP datasets.
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Figure 3—figure supplement 1—source data 1
PDF file containing original western blots and agarose gel of libraries for Figure 3—figure supplement 1A and B, indicating the relevant bands and conditions.
- https://cdn.elifesciences.org/articles/104808/elife-104808-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original files for western blot and agarose gel of libraries displayed in Figure 3—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/104808/elife-104808-fig3-figsupp1-data2-v1.zip
Figure 4 with 2 supplements
Differential gene expression and alternative exon incorporation rates in Rbm20 conditional knockout cells.
(A) Volcano plot of differential gene expression in RiboTrap-isolated mRNAs from Rbm20WTvs. Rbm20ΔSlc17a6 olfactory bulb (P35). Significantly regulated genes shown in red, cutoff fold change (FC) of 1.5, adjusted p-value<0.01, total number of up- and downregulated noted on top. Note that Rbm20 itself is strongly reduced, outside the axis limits, and not represented in this plot (see Supplementary file 4). (B) Volcano plot of the differential gene expression in RiboTrap-isolated mRNAs from Rbm20WT vs. Rbm20ΔPV mouse neocortex (P35) as in panel A. The Y-chromosomal genes Ddx3y, Uty, Kdm5d, Eif2s3y were highly differentially expressed due to the larger number of Rbm20 mutant males used in the Ribotrap isolations (3 wild-type females and 1 wild-type male vs. 4 knockout male mice were used for this experiment). These genes and Rbm20 itself were excluded from the plot (see Supplementary file 4 for complete data). (C) Volcano plot representing differentially included exons in Rbm20ΔSlc17a6 RiboTrap-isolated mRNAs from olfactory bulb neurons. The dotted lines correspond to FC values of 1.5 and –1.5 and –log10 (p-value) of 1.3. Significantly regulated exons (FC 1.5 and p<0.05) are shown in red. (D) Volcano plot representing differentially included exons in Rbm20ΔPV RiboTrap-isolated mRNAs from cortical interneurons. The dotted lines correspond to FC values of 1.5 and –1.5 and –log10 (p-value) of 1.3. Significantly regulated exons (FC 1.5 and p-value<0.05) are shown in red. (E) Number of exons differentially expressed in Slc17a6+ cells isolated from the olfactory bulb of Rbm20ΔSlc17a6 mice and number of exons with significant RBM20 CLIP peaks in binding sites within indicated distances. Equivalent information is provided for exons divided by annotations for specific features of regulation: alternative polyadenylation (last exons), alternative transcription start sites (TSS), complex events, and cassette exons.
Figure 4—figure supplement 1
Normal morphological differentiation of mitral cells in the absence of RBM20.
(A, B) Immunostaining of RBM20 (green), RPL22-HA (gray), and DAPI (blue) in the cortex and olfactory bulb of P35 PvalbCre::Rpl22HA/HA and (B) Slc17a6Cre::Rpl22HA/HA mice, compared to corresponding littermates carrying the conditional Rbm20fl/fl alleles (Rbm20ΔPV and Rbm20ΔSlc17a6, lower panels). (C) RBM20-positive (green) mitral cells retrogradely labeled through injection of rAAV2-SYN-Cre virus (red) in piriform cortex of Rosa26LSL-tdTomato mice at P35. Scale bar: 100 µm. (D) Schematic illustration of the rAAV2-DIO-eGFP virus injection into the piriform cortex of Slc17a6Cre mice for retrograde labeling of olfactory bulb mitral cells. Representative image of the site of viral injection in the piriform cortex. Scale bar: 500 µm. (E) GFP+ mitral cell (gray) retrogradely labeled through injection of rAAV2-SYN-DiO-GFP virus in the piriform cortex of Slc17a6Cre and Slc17a6Cre::Rbm20fl/fl (Rbm20ΔSlc17a6) mice. Tracing of the neuronal arborization is displayed on the right. Scale bar: 500 µm. (F) Quantification of the mean number of branches calculated per animal and the mean length of the neuronal tufts in reconstructed mitral cells of Slc17a6Cre and Rbm20ΔSlc17a6 mice. n=4.
Figure 4—figure supplement 2
Quality control analysis of Ribo-TRAP RNA-sequencing samples.
(A–B) Fold enrichment (FC) of markers specific to inhibitory cortical neurons or glutamatergic neurons for wild-type (WT) and conditional knockout (cKO) samples. For PV samples, the following markers were tested: Pvalb, Gad67, Slc32a1, Slc17a7, Gfap. For glutamatergic: Slc17a7, Slc17a6, Gad67, Pcdh21, Gfap, Slc32a1, and Tbr2. (C) Coverage plot indicating the percentage of read bases at a given position of the transcript. No sample displayed 3’ or 5’ coverage bias across the transcript length. (D) Bar plot of each biological replicate where for each sample the following parameters are indicated: the proportion of number of reads uniquely mapped, mapped to multiple loci, mapped to too many loci, or unmapped reads for all the samples. All samples show highly similar values across biological replicates, as well as across brain region and genotype, suggesting a high consistency and homogeneity of the RNA-sequencing data. (E) Bar plot representing the relative percentage of reads falling on genomic features for all the biological samples. (F) PCA of genes expressed in each olfactory bulb sample (n=5 biologically independent samples per genotype). Variance explained by the principal components 1 and 2 (PC1 and PC2) is indicated. Gene expression values were normalized by variance stabilizing transformation (VST).
Figure 5
Long pre-mRNAs are depleted in Rbm20ΔSlc17a6 mitral cells.
(A) Metagene coverage plots of CLIP peaks across all RBM20-bound introns. Peak density across exons from the same transcripts is shown for comparison. (B) Total number of genes up- or downregulated (FC>1.5 and adjusted p-value<0.01) in glutamatergic cells from the olfactory bulb of Rbm20ΔSlc17a6 mice. The fraction of genes with significant RBM20 CLIP peaks is indicated in blue. (C) Illustration of gene ontologies enriched amongst up- and downregulated genes (cellular component analysis with Bonferroni correction [p-value 0.05]). Minimum number of five genes identified per category. (D) Correlation of differential gene expression in Rbm20ΔSlc17a6 cells, intron length, and CLIP-seq data. Genes were ranked by FC in differential gene expression and mean total intron length (left), and mean number of intronic CLIP peaks (right) for blocks of 100 genes were plotted. Ranks of the genes meeting FC cutoff for down- or upregulation are highlighted in color. Spearman’s coefficients and p-values are indicated. (E) Boxplot showing the total intron length per gene (expressed in log10 scale) for categories of downregulated genes (all, with or without RBM20-binding sites), nonregulated genes, and upregulated genes in Rbm20ΔSlc17a6 in RiboTrap isolates from the olfactory bulb. Only annotated genes are plotted (number of genes shown at the bottom). p-Values from the Wilcoxon test are indicated. Medians: all downregulated genes 83.2 kb, downregulated genes with peaks: 152.4 kb; downregulated genes without peaks: ~33.6 kb; all non-regulated genes: 21.5 kb; all upregulated genes: 7.4 kb. (F) Boxplot (log10 scale) illustrating the total length of introns found in genes identified in our RBM20 Ribo-TRAP dataset (gray) compared to introns presenting RBM20-binding sites (green). RBM20-bound introns exhibit a higher intron length. p-Values from the Wilcoxon test are indicated. Medians: expressed introns: 1.4 kb; introns with peaks: 30.5 kb. (G) Plot representing the cumulative probability distribution of intron length between the two groups of introns as in panel F. p-Value (Kolmogorov-Smirnov test) is indicated.
Additional files
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Supplementary file 1
Identified RBM20-binding sites in the heart and olfactory bulb tissues.
List of RBM20 peaks identified on transcript mRNAs in the heart and in olfactory bulb tissues. Peaks were identified through the peak caller Clipper followed by irreproducible discovery rate (IDR) analysis between replicates. In this table, beyond the standard output parameters produced by IDR, columns containing information about the annotation of the targeted transcript and the position of RBM20-binding site in relation to the exon-intron boundaries (R package ‘AnnotatR’) are reported. Moreover, the list of read counts summarized using featureCounts for identification of expressed genes in the input samples of heart and olfactory bulb is reported.
- https://cdn.elifesciences.org/articles/104808/elife-104808-supp1-v1.xlsx
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Supplementary file 2
Gene ontology analysis of transcripts directly bound by RBM20.
Gene ontology analysis results by Panther of mRNA transcripts directly bound by RBM20 RNA-binding protein in both heart and olfactory bulb tissues.
- https://cdn.elifesciences.org/articles/104808/elife-104808-supp2-v1.xlsx
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Supplementary file 3
Expressed genes and percentage of mapped and unmapped unique reads in Ribo-TRAP RNA-sequencing experiments.
Read counts summarized using featureCounts. For each sample from either Slc17a6+ neurons of the olfactory bulb or PV+ interneurons of the neocortex.
- https://cdn.elifesciences.org/articles/104808/elife-104808-supp3-v1.xlsx
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Supplementary file 4
Summary of differential gene expression analysis.
Expression values (FPKM) of genes identified in Parvalbumin-positive and Slc17a6+ neurons in Ribo-TRAP RNA-sequencing experiments and the results of their differential gene expression analysis by DESEQ2 in wild-type vs. Rbm20 conditional knockout mice.
- https://cdn.elifesciences.org/articles/104808/elife-104808-supp4-v1.xlsx
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Supplementary file 5
Summary of alternative exon usage analysis.
Expression values (RPKM) of all the exons identified in Parvalbumin-positive and Slc17a6-positive cells in Ribo-TRAP RNA-sequencing experiments and the results of the alternative exon usage analysis in wild-type vs. Rbm20 conditional knockout mice.
- https://cdn.elifesciences.org/articles/104808/elife-104808-supp5-v1.xlsx
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Supplementary file 6
Gene ontology of deregulated transcripts and alternatively spliced exons.
Gene ontology analysis results by Panther for deregulated transcripts (all, downregulated, and upregulated) and exon usage analysis in olfactory bulb neurons upon RBM20 ablation.
- https://cdn.elifesciences.org/articles/104808/elife-104808-supp6-v1.xlsx
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Supplementary file 7
Analysis of intron length.
Analysis of intron length in deregulated, nonregulated, and RBM20-bound transcripts in olfactory bulb neurons.
- https://cdn.elifesciences.org/articles/104808/elife-104808-supp7-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/104808/elife-104808-mdarchecklist1-v1.pdf
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Dilated cardiomyopathy-associated RNA-binding motif protein 20 regulates long pre-mRNAs in neurons
eLife 14:RP104808.
https://doi.org/10.7554/eLife.104808.3
