SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition

Abstract

Mutations affecting spliceosomal proteins are the most common mutations in patients with myelodysplastic syndromes (MDS), but their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2's normal sequence-specific RNA binding activity, thereby altering the recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2, which triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in the splicing of key regulators, and impaired hematopoiesis.

Figure 1. Conditional expression of Srsf2P95H results in myeloid dysplasia, a phenotype distinct from heterozygous or homozygous loss of Srsf2

(A) Depiction of the Srsf2P95H allele. (B) RNA-seq of LSK cells in Mx1-cre Srsf2WT and Mx1-cre Srsf2 P95H/WT mice. (C) White blood cell (WBC) count, (D) hemoglobin (Hb), and (E) mean corpuscular volume (MCV) of red blood cells of CD45.1 recipient mice 18 weeks following noncompetitive transplantation of bone marrow from CD45.2+ Mx1-cre Srsf2WT, Mx1-cre Srsf2fl/WT, Mx1-cre Srsf2fl/fl, and Mx1- cre Srsf2 P95H/WT mice (n=10 mice/genotype for all genotypes except Mx1-cre Srsf2fl/WT where n=5; pIpC was administered to recipient mice 4 weeks following transplantation). (F) H&E staining of femurs (Bars: 50 µm) and (G) peripheral blood smears from Mx1-cre Srsf2WT, Mx1-cre Srsf2fl/fl or Mx1-cre Srsf2 P95H/WT mice (Bars: 10 µm). A representative neutrophil (left) and erythroid precursor (right) is shown for Srsf2 WT and KO mice. Mx1-cre Srsf2P95H cells were marked by hypolobated and hypogranulated neutrophils (left 2 photos) and nuclear irregularities as well as cytoplasmic vacuolization and blebbing of erythroid precursors (2 rightmost photos). Error bars represent mean ± SD; ***p < 0.001; ****p < 0.0001. See also Figure S1.

Figure 2. Conditional expression of Srsf2P95H results in expansion of hematopoietic stem and progenitor cells with increased cell proliferation and apoptosis

(A) Enumeration and (B) FACS analysis of BM LSK cells, long-term hematopoietic stem cells (LT-HSC), restricted hematopoietic progenitor cell fractions 1 (HPC-1) and 2 (HPC-2) and multipotent progenitor (MPP) cells (Oguro et al., 2013) in 12-week old Mx1-cre Srsf2 WT and Mx1-cre Srsf2 P95H/WT mice (n=5 mice/genotype). (C) Cell cycle analysis of LSK cells from Mx1-cre Srsf2WT or Mx1-cre Srsf2 P95H/WT mice with in vivo BrdU administration. Representative FACS plot analysis showing gating on LSK cells followed by BrdU versus DAPI stain is shown on the left. (D) Relative quantification of the percentage of LSK cells in S, G2M, and G1 phase is shown on the right (n=8 mice per group). (E) Relative quantification of the percentage of Annexin V+/DAPI- LSK cells (n=8 mice/genotype). Error bars represent mean ± SD; *p < 0.05; **p < 0.01; ****p < 0.0001. See also Figure S2.

Figure 3. Srsf2P95H mutation impairs hematopoietic stem cell self-renewal in a manner distinct from Srsf2 loss

(A) Depiction of competitive bone marrow (BM) transplantation assay. (B) Percentage of CD45.2+ chimerism in the peripheral blood of recipient mice (n=10 mice/genotype). (C) Chimerism and (D) flow cytometric enumeration of CD45.2+ LSK (left) and myeloid progenitor (MP; lineage-negative Sca1-c-Kit+) (right) cells in BM of Mx1-cre Srsf2WT, Mx1-cre Srsf2fl/WT, Mx1-cre Srsf2fl/fl and Mx1-cre Srsf2 P95H/WT mice 14 weeks after pIpC injection. Error bars represent mean ± SD; **p< 0.001, ***p < 0.0002; ****p< 0.0001. See also Figure S3.

Figure 4. SRSF2 mutations alter exonic splicing enhancer preference

(A) Scatter plot of cassette exon inclusion in K562 cells expressing empty vector or SRSF2 P95R. Percentages, percent of alternatively spliced cassette exons with increased or decreased inclusion. Red and blue dots represent individual cassette exons that are promoted or repressed in SRSF2 P95R versus empty vector cells, respectively. Promoted and repressed cassette exons are defined as those whose inclusion levels are increased or decreased by ≥10% with a Bayes factor ≥5, as estimated by Wagenmakers’s framework (Wagenmakers et al., 2010). (B) Enriched (right) and depleted (left) k-mers in cassette exons promoted versus repressed in SRSF2 P95R versus WT cells. (C) Scatter plot of cassette exon inclusion in TF-1 cells following transfection with a siRNA against SRSF2 or a control non-targeting siRNA (“KD”, knockdown). Percentages, percent of alternatively spliced cassette exons with increased or decreased inclusion. (D) Enriched (right) and depleted (left) k-mers in cassette exons promoted versus repressed in SRSF2 KD versus control cells. (E) Mean enrichment of all variants of the SSNG motif in cassette exons promoted versus repressed in TF-1 cells following SRSF2 knockdown and K562, LSK, and MP cells expressing WT or mutant SRSF2. Error bars, 95% confidence intervals estimated by bootstrapping. (F) Relative frequency of CCNG and GGNG motifs in cassette exons promoted versus repressed by SRSF2 mutations in LSK and MP cells (top), K562 cells (left), and primary AML and CMML samples with or without SRSF2 mutations (right; sample numbers correspond to patient identifiers in Table S1). Shading, 95% confidence interval by bootstrapping. The cartoon illustrates a portion of a meta-gene containing the differentially spliced cassette exon; from left to right, the features are the upstream exon (gray box) and intron (black line), the cassette exon (black box, vertical dashed lines), and the downstream intron (black line) and exon (gray box). Horizontal axis, genomic coordinates defined with respect to the 5' and 3' splice sites, where 0 is the splice site itself. Vertical axis, relative frequency of the indicated motifs over genomic loci containing cassette exons promoted versus repressed by SRSF2 mutations (log scale). See also Figure S4.

Figure 5. Proline 95 mutations change RNA-binding specificity of the SRSF2 RNA recognition motif domain (RRM) in vitro and lead to relocation of the N- and C-termini

(A) ITC raw data and binding curve for SRSF2 RRM P95H mutant with 5’-uCCAGu-3’ and 5’-uGGAGu-3’ RNA. (B) Change in RNA-binding affinity (%) for SRSF2 RRM P95H (blue), P95L (green) and P95R (black) mutants compared to WT (red) (Daubner et al., 2012), using RNA targets 5’-uCCAGu-3’, 5’-uGCAGu-3’, 5’-uCGAGu-3’ and 5’-uGGAGu-3’. (C) Change in RNA-binding specificity of SRSF2 RRM WT, P95H, P95L and P95R with 5’-UCCAGU-3’ (blue), 5’- UGCAGU-3’ (dark grey), 5’-UCGAGU-3’ (light grey) and 5’-UGGAGU-3’ RNA (orange). (D) (left) Overlay of 2D [¹⁵N-¹H] HSQCs of wild type (red) and P95H mutant (blue) bound to 5’-UCCAGU-3’ RNA, with negative peaks in green (WT) and light-green (mutant). (right) Difference of the chemical shift perturbations of P95H mutant and wild type. Positive values (blue) with a higher perturbation with the P95H mutant and negative values (red) with a higher perturbation with the WT are shown. Missing assignments are marked with grey bars and proline with a grey P. Residues with the highest difference are depicted in both graph and spectra. See also Figure S5.

Figure 6. SRSF2-mutant primary murine and patient samples exhibit convergent splicing alterations

(A) Intersection of genes exhibiting differential splicing in SRSF2 mutant versus WT mouse LSK and MP cells and primary AML and CMML samples (restricted to orthologous genes). (B) IGV/sashimi plot illustrating the EZH2 cassette exon promoted by SRSF2 mutations in multiple datasets analyzed here (top; patient numbers listed in the Sashimi plot correspond to numbers in Table S1 detailing patient characteristics). The DNA sequence conservation of the locus, as estimated by phastCons (Siepel et al., 2005), across 30 vertebrate species is shown in the track below the Sashimi plot. (C) Bar plot describing the percentage of EZH2 transcripts harboring a specific cassette exon in SRSF2 mutant relative to WT primary AML samples from RNA-seq data. Error bars, 95% confidence intervals. (D) RT-PCR of EZH2 exon inclusion event in an independent set of SRSF2 WT and mutant AML samples. (E) qRT-PCR of EZH2 inclusion isoform in SRSF2 P95H mutant cell line K052 cells with or without UPF1 knockdown and actinomycin D treatment. (F) Western blot (WB) analysis for EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) in SRSF2/EZH2 WT (TF-1, K562) and SRSF2 P95H mutant/EZH2 WT (K052) AML cell lines. (G) WB analysis for EZH2, H3K27me3, and FLAG epitope in K562 cells with lentiviral overexpression of N-terminal FLAG tagged SRSF2 WT, SRSF2P95H, SRSF2 P95L, or SRSF2 P95R (left). Relative quantification of EZH2 protein expression by WB to total histone H3 expression in K562 cells expressing SRSF2 mutants relative to WT is shown on right. (H) EZH2 and SRSF2 mutations are mutually exclusive in sequencing of >1,000 MDS patients (Bejar et al., 2012; Ernst et al., 2010; Haferlach et al., 2014; Muto et al., 2013; Papaemmanuil et al., 2013). (I) Cartoon of EZH2 cassette exon, with SSNG motifs highlighted and mutations to GG equivalents shown. (J) EZH2 cassette exon inclusion for minigenes containing the endogenous cassette exon or a cassette exon with mutation of motifs 1, 2, and/or 3 to the GG equivalent. (K) Photographs (left) and enumeration (right) of c-Kit+/ZsGreen1+ cells from Srsf2 WT or Srsf2P95H mice 14 days after overexpression of empty vector or EZH2 cDNA and plating in methylcellulose media. See also Figure S6 and Table S1–S5.