SRSF2 Mutations Impair Hematopoietic Differentiation By Altering Exonic Splicing Enhancer Preference

Spliceosomal mutations account for the most frequent class of mutations in patients with MDS and CMML, yet the mechanism by which these mutations perform their driver function is not well understood. Moreover, no genetically engineered murine models for expression of spliceosomal gene mutations from their endogenous loci have been reported. Given the genetic heterogeneity of primary patient samples, we generated a model for conditional expression of the commonly occurring SRSF2P95H mutation from the endogenous murine locus of Srsf2. We compared expression of the Srsf2P95H mutation with genetic inactivation of 0, 1 or 2 copies of Srsf2 to understand the biological and mechanistic consequences of spliceosomal mutations relative to loss-of-function.

Mx1-cre Srsf2P95H/wildtype mice exhibited significant morphologic dysplasia, leukopenia, macrocytic anemia, and preserved bone marrow (BM) cellularity as early as 2 weeks after mutation expression (Figure A). Moreover, Mx1-cre Srsf2P95H/wildtype mice exhibited an increase in hematopoietic stem/progenitor cells (HSPCs) with an increase in lineage-negative Sca1+ c-Kit+ cells (LSK cells) in S-phase and early apoptosis. In competitive transplantation, Srsf2P95H mice HPSCs were expanded in the BM at 16 weeks post-transplantation despite having a reduced contribution to peripheral blood chimerism. In contrast, mice with homozygous deletion of Srsf2 exhibited anemia and leukopenia due to BM aplasia with striking loss of HSPCs (Figure B). Collectively, these data show that Srsf2 is required for hematopoiesis, while mutations in Srsf2 provide a competitive advantage at the level of HSPCs but impair differentiation into mature circulating blood elements.

Next, to identify transcriptional and post-transcriptional alterations caused by SRSF2 mutations, we performed deep RNA-seq on sorted HSPC populations from wildtype and Srsf2P95H mice, stable K562 cell lines ectopically expressing an empty vector or a single allele of SRSF2 (WT, P95H, P95L, P95R), as well as primary CMML and AML patient samples. We quantified global changes in splicing of ~125,000 alternative splicing events and ~160,000 constitutive splice junctions associated with SRSF2 mutations in these datasets. Intersection of differentially spliced genes in primary murine HSPC, CMML, and AML samples identified 75 genes that were differentially spliced in association with SRSF2 mutations in murine HSPCs and at least one primary patient cohort. Many of the genes that were differentially spliced in SRSF2 mutant cells participate in biological processes of known importance in myeloid malignancies. For example, a cassette exon of EZH2 that alters the reading frame likely to induce nonsense-mediated decay was promoted by SRSF2 mutations, while a frame-preserving cassette exon of BCOR was repressed by SRSF2 mutations.

We next sought to determine how SRSF2 mutations alter SRSF2Õs normal role in RNA splicing. As SRSF2 recognizes exonic splicing enhancer (ESE) elements within the pre-mRNA to promote exon recognition, we hypothesized that SRSF2 mutations might alter its normal sequence-specific activity. To test this, we performed an ab initio motif identification screen to identify motifs that were enriched or depleted in cassette exons promoted versus repressed in primary Srsf2P95H cells relative to wildtype. This analysis identified CCAG and GGTG as the most enriched and depleted consensus motifs, respectively. A recent solution structure of SRSF2 in complex with RNA revealed that SRSF2 normally recognizes the motifs CCNG and GGNG equally well. Analysis of the spatial distribution of CCNG and GGNG motifs across genomic loci containing cassette exons that were promoted or repressed in association with SRSF2 mutations revealed that CCNG and GGNG were respectively enriched and depleted specifically over the cassette exons, and not over the flanking introns or exons, that were differentially spliced in association with SRSF2 mutations (Figure C). Together, our data indicate that mutant SRSF2 drives widespread changes in splicing due to alterations in its sequence-specific recognition of exonic splicing enhancers.

The biological as well as molecular data here identify an effect of the SRSF2P95H mutation distinct from haploinsufficient or complete loss of SRSF2 and reveal that mutations in SRSF2 alter ESE preference to contribute to key aspects of MDS.

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