| Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders that are characterized by ineffective hematopoiesis, deficiencies in circulating erythrocytes, neutrophils, and/or thrombocytes, and a high risk for the development of acute myeloid leukemia (AML). De novo MDS is an age related disorder with the risk of MDS greatly increasing past the age of 60. In addition to de novo MDS, MDS can also occur as a result of treatment for previous cancer, such as breast cancer, Hodgkin's lymphoma, or non-Hodgkin's lymphoma. Nearly 30% patients progress to AML within a year of diagnosis, and nearly 60% of MDS patients do not survive more than 3 years past diagnosis, regardless of progression to AML. However, the cause of MDS is still unknown, and there are few effective treatments. Whole exome sequencing has revealed that nearly 50% of MDS patients harbor somatic mutations in proteins involved in pre-mRNA 3' splice site recognition in their bone marrow. These mutations are most common in the genes U2AF1, SRSF2, SF3B1, and ZRSR2.;SRSF2 is a member of the SR protein family of splicing regulators. These proteins commit the pre-mRNA to splicing through their interactions with members of the core spliceosome. The structure of SRSF2 consists of an RNA recognition motif and an arginine-serine rich domain involved in protein-protein interactions. These regions are separated by a hinge region of approximately 12 amino acids. In addition, there is a nuclear retention signal in the C-terminal end of the protein that prevents SRSF2 from shuttling to the cytoplasm, unlike other SR proteins that participate in mRNA export. In addition to SRSF2's function in constitutive and alternative splicing, SRSF2 is also required for an RNA-dependent regulation of transcriptional elongation at many highly regulated gene promoters.;The most common mutation of SRSF2 in MDS patients is a missense mutation that alters proline at position 95 in the hinge region to histidine, leucine, or arginine. These mutations occur in ∼15% of MDS patients. The narrow and specific nature of these mutations, along with their presence in the earliest stages and persistence throughout the disease, suggest an important, yet unidentified, role in MDS pathogenesis. While recent research has shown that these MDS-associated SRSF2 mutants have different RNA binding preferences than wildtype SRSF2, their role in specific alternative splicing events has yet to be elucidated.;In this study, we developed stable inducible cell lines in TF-1 erythroleukemia cells that express either wildtype SRSF2, SRSF2 with proline 95 point mutations found in MDS, or SRSF2 with a deletion of one of the 4 major domains of the protein. Analysis of these cell lines showed that the MDS-associated SRSF2 mutants have similar expression levels, subcellular localization, and total phosphorylation levels as wildtype SRSF2. However, cells expressing mutant SRSF2 exhibited higher levels of apoptosis than wildtype SRSF2. Alternative splicing analysis showed similar function for wildtype and the MDS-associated SRSF2 mutants in the splicing of the endogenous 3'UTR of SRSF2. However, a unique alternative splicing event for the SRSF2 mutants was observed for CDC25C. Specifically, an increase in the C5/C1 isoform ratio was observed only in cells expressing MDS-associated SRSF2 mutants, but not in cells expressing wildtype SRSF2 or SRSF2 deletion mutants. This splicing event is similar to the CDC25C alternative splicing event observed after treatment of cells with drugs that induce the DNA damage response. However, induction of the DNA damage response is not required for alternative splicing of CDC25C in SRSF2 mutant cell lines. |
