Rbm3 deficiency leads to transcriptome-wide splicing alterations

Abstract

Rbm3 (RNA-binding motif protein 3) is a stress responsive gene, which maintains cellular homeostasis and promotes survival upon various harmful cellular stimuli. Rbm3 protein shows conserved structural and molecular similarities to heterogeneous nuclear ribonucleoproteins (hnRNPs), which regulate all steps of the mRNA metabolism. Growing evidence is pointing towards a broader role of Rbm3 in various steps of gene expression. Here, we demonstrate that Rbm3 deficiency is linked to transcriptome-wide pre-mRNA splicing alterations, which can be reversed through Rbm3 co-expression from a cDNA. Using an MS2 tethering assay, we show that Rbm3 regulates splice site selection similar to other hnRNP proteins when recruited between two competing 5${}^{\text{'}}$ splice sites. Furthermore, we show that the N-terminal part of Rbm3 encompassing the RNA recognition motif (RRM), is sufficient to elicit changes in splice site selection. On the basis of these findings, we propose a novel, undescribed function of Rbm3 in RNA splicing that contributes to the preservation of transcriptome integrity.

Keywords: RNA-seq; Rbm3; isoform switch; splicing; transcriptional fidelity.

Figure 1.

Rbm3 deficiency alters splicing landscape genome-wide. (A) homology between the Glycine-rich domain (GRD) and RNA recognition motif (RRM) of Rbm3 and the two closest hnRnps: hnRNP A0 and Rbmx. (B) graphical representation and quantification of significant as events and affected genes detected by rMATS. SE: skipped exon; A5SS: alternative 5$\mathrm{\prime }$splice site; A3SS: alternative 3$\mathrm{\prime }$splice site; MXE: mutually exclusive exons; RI: retained intron. (C) number of overlapping genes in each as event category. (D) heat map showing the altered splicing landscape of AS genes. Red colour represents increased, while blue colour represents decreased sequencing read inclusion/exclusion ratios (calculated from rMATS Ψ- values). Each column represents a biological replicate. For example, in the SE event box, negative (blue) Ψ- values represent lower exon inclusion frequency in KO cells, indicating that exon skipping is frequent in Rbm3^(def) cells. (E) gene ontology analysis showing biological processes significantly enriched with AS genes.

Figure 2.

Molecular consequences of Rbm3 deficiency. (A) table containing the detected type and number of transcript isoform switches on mRNA level (green table) and their predicted consequences on their corresponding protein structure (blue table) and subcellular localization (yellow table) in Rbm3^(def) L929 cells. Abbreviations: untranslated regions (UTR), open reading frame (ORF), nonsense-mediated decay (NMD), transcription start site (TSS), intrinsically disordered region (IDR). Semi-quantitative RT-PCR images and quantifications confirm increased exon skipping in candidate genes: Map3k7 (B), Cdk7 (C), Cdk4 (D) and Clk2 (E). Top left panel: graphical representation of transcript isoform structures (thick black bars are exons, thin lines are introns, black arrows show strand directionality and purple arrows show oligo locations for PCR validation). Top right panels: RT-PCR images and quantification of the detected exon-skipping events. Data represent means of the exon inclusion-to-skipping ratio ± standard deviation from three independent biological replicates. Statistical significance was calculated using an unpaired two-tailed Student’s t-test. ** = p < 0.01; **** = p < 0.0001.

Figure 3.

Rbm3 co-expression restores exon-inclusion rates. (A – F) RT-PCR analysis and quantification of exon skipping events in Rbm3^(wt), Rbm3^(def), and in Rbm3^(def) L929 cells where Rbm3-gfp is ectopically overexpressed. Genes are: Map3k7 (A), Cdk7 (B), Cdk4 (C) and Clk2 (D). Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent standard deviation from biological triplicates. * = p < 0.05; ** = p < 0.01; n.S. = not significant. Eif3f (E) and gapdh (F) are negative control genes without detectable as events in Rbm3^(def) L929 cells.

Figure 4.

Characterization of mis-spliced genes upon Rbm3 deficiency. (A – C) gene ontology analysis showing biological processes significantly enriched with as genes in RNA-seq data from Rbm3-deficient activated mouse lung immune cells (A), mouse hippocampus (B) and in human pancreatic cancer cells (C). (D – F) relative frequency plots of as genes (blue line) and all expressed genes (black line) in Rbm3^(def) L929 cells, lung immune cells and mouse hippocampus dataset for the comparison of the following characteristics: (D) gene expression level (log2 scale), (E) gene length (log10 scale) and (F) exon counts (log2 scale). Wilcoxon rank-sum test, **** represent the following p-values from left to right panels: (D) p < 2.2 × 10⁻¹⁶, p < 2.2 × 10⁻¹⁶, p = 5.648 × 10⁻¹², p = 1.617 × 10⁻¹¹; (E) p < 2.2 × 10⁻¹⁶, p < 2.2 × 10⁻¹⁶, p < 2.2 × 10⁻¹⁶, p < 2.2 × 10⁻¹⁶; (F) p < 2.2 × 10⁻¹⁶, p = 1.39 × 10⁻¹³, p = 1.568 × 10⁻⁵ and p = 6.987 × 10⁻⁷.

Figure 5.

Rbm3 alters splice-site selection. (A) graphical representation of the splicing reporter assay which consists of two main components: 1.) Rbm3 (full protein, or RRM or GRD separately) is tagged with the MS2 bacteriophage protein that recognizes and binds to bacterial stem-loop RNA structures. 2.) an artificial mRNA containing stem-loop structures in between two competing (upstream and downstream) splice sites. MS2 tethering of potential splicing regulators could edit various mRNA sizes: splicing repressors cause intron retention, producing a 433 bp fragment with the oligos targeting the exons (grey bars), while splicing regulators with preferential 5$\mathrm{\prime }$splice site recognition might produce either a 238 bp fragment (SD1 down) or a 138 bp fragment (SD1 upstream). (B) RT-PCR results of our assay show increased use of the SD1 upstream 5$\mathrm{\prime }$SS when Rbm3 is bound, indicating that Rbm3 is a splicing regulator. (C) bar diagram showing the quantification of the 5$\mathrm{\prime }$splice site usage. (n = 5 biological replicates, one-way ANOVA followed by post-hoc Tukey’s test, p < 0.01) (D) and (E) PCR assay and quantification of 5$\mathrm{\prime }$splice site usage in the presence of ectopically expressed Rbm3-gfp without MS2 tag (n = 4 biological replicates, one-way ANOVA followed by post-hoc Tukey’s test).