Myelodysplastic Syndromes (MDS) are a heterogeneous group of clonal disorders characterized by dysplastic hematopoietic cells, ineffective hematopoiesis, and a variable risk of transformation to acute myeloid leukemia (AML) with few curative therapeutic options. Recent studies have established that genes encoding spliceosomal proteins are the most common mutational targets in MDS, with ~60% of MDS patients harboring a spliceosomal gene mutation. These spliceosomal mutations occur exclusively in a heterozygous context, are mutually exclusive with one another, and frequently co-occur with mutations affecting specific epigenetic regulators. However, the molecular and biological reasons for this distinctive mutational pattern are not known, nor is it understood how spliceosomal mutations contribute to dysplastic hematopoiesis. Here, we propose to determine the genetic basis as well as mechanistic and phenotypic consequences of mutations affecting the spliceosomal gene SRSF2. Our team consists of a physician-scientist with experience in MDS biology and patient care (Abdel-Wahab) and a basic scientist with expertise in RNA splicing and genomics (Bradley). In preliminary studies, we found that SRSF2 mutations result in impaired hematopoiesis and altered exon recognition, and we generated extensive novel reagents to explore the functional importance of SRSF2 mutations. We propose to build on these preliminary studies as follows: Aim 1, Determine how SRSF2 mutations alter SFSF2's normal role in pre-mRNA splicing to promote myelodysplasia; Aim 2, determine the biological and molecular basis for the association between SRSF2 mutations and additional genetic alterations; Aim 3, Identify and test therapeutic strategies for targeting malignant cells with SRSF2 mutations. The significance of these studies is that they will definitively connect SRSF2 mutations to altered RNA splicing mechanisms and dysplastic hematopoiesis. The health relatedness is that our studies may identify new therapeutic opportunities for MDS by selectively targeting cells carrying SRSF2 mutations.