Dilated cardiomyopathy-associated RNA Binding Motif Protein 20 regulates long pre-mRNAs in neurons

Giulia Di Bartolomei
Raul Ortiz
Dietmar Schreiner
Susanne Falkner
Esther E Creemers
Peter Scheiffele author has email address

Biozentrum, University of Basel, Basel, Switzerland
Department of Experimental Cardiology, Amsterdam Cardiovascular Sciences, Amsterdam UMC, location University of Amsterdam, Amsterdam, Netherlands

https://doi.org/10.7554/eLife.104808.2

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Figures and data

Characterization of Rbm20 expression in the brain
A. Dot plot of the expression of a hand-curated list of 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 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 (grey). 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 Vglut2^Cre :: Rpl22^HAmouse 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 Rbm20^KO 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 (for GAPDH detection).
F. Immunofluorescence of RBM20 (green) expression in the mitral cell layer (MCL) of P0 and P35 Tbx21^Cre::Ai9^tdTomatomice. Mitral cells and tufted neurons are labelled with the tdTomato reporter (red). Scale bar 100 µm.

Identification of RBM20 direct targets in the heart and olfactory bulb
A. Sub-nuclear localization of RBM20 (green) in heart cardiomyocytes (left) and mitral cells of the olfactory bulb (right) of 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 poly-adenylation signal (up). Schematic representation of the resulting RBM20^HA 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 Rbm20^HAtagged mice. GAPDH is used as a loading control.
D, E (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, non-coding 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.

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 grey). CLIP peaks, considered statistically significant by 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 TableS1 for coordinates.
B. Illustration of the 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: 5 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: 5 genes.

Differential gene expression and alternative exon incorporation rates in Rbm20 conditional knock-out cells
A. Volcano plot of differential gene expression in RiboTrap-isolated mRNAs from Rbm20^WT vs. Rbm20^ΔVglut2 olfactory bulb (P35). Significantly regulated genes shown in red, cut-off fold-change (FC) of 1.5, adjusted p-value <0.01, total number of up- and down-regulated noted on top. Note that Rbm20 itself is strongly reduced, outside the axis limits, and not represented in this plot (see Table S4).
B. Volcano plot of the differential gene expression in RiboTrap-isolated mRNAs from Rbm20^WT vs. Rbm20^ΔPV 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 knock-out male mice were used for this experiment). These genes and Rbm20 itself were excluded from the plot (see Table S4 for complete data).
C. Volcano plot representing differentially-included exons in Rbm20^ΔVglut2 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 Rbm20^ΔPV 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 Vglut2⁺ cells isolated from the olfactory bulb of Rbm20^ΔVglut2 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 poly-adenylation (last exons), alternative transcription start sites (TSS), complex events and cassette exons.

Long pre-mRNAs are depleted in Rbm20^ΔVglut2 mitral cells
A. Metagene coverage plots of CLIP peaks across all RBM20-bound introns. Peak density across exons from the same transcripts are shown for comparison.
B. Total number of genes up- or down-regulated (FC > 1.5 and adjusted p-value <0.01) in glutamatergic cells from the olfactory bulb of Rbm20^ΔVglut2 mice. The fraction of genes with significant RBM20 CLIP peaks is indicated in blue.
C. Illustration of gene ontologies enriched amongst up- and down-regulated genes (cellular component analysis with Bonferroni correction (p-value 0.05). Minimum number of 5 genes identified per category.
D. Correlation of differential gene expression in Rbm20^ΔVglut2 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 cut-off for down- or up-regulation 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), non-regulated genes, and up-regulated genes in Rbm20^ΔVglut2 in RiboTrap isolates from the olfactory bulb. Only annotated genes are plotted (number of genes shown at the bottom). P-values from Wilcoxon test are indicated. Medians: all down-regulated genes 83.2 kb, down-regulated genes with peaks: 152.4 kb; down-regulated genes without peaks: ∼33.6 kb; all non-regulated genes: 21.5 kb; all up-regulated genes: 7.4 kb.
F. Boxplot (log₁₀ scale) illustrating the total length of introns found in genes identified in our RBM20 Ribo-TRAP dataset (grey) compared to introns presenting RBM20 binding sites (green). RBM20 bound introns exhibit a higher intron length. P-values from 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.

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