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January Graduate Student Research Forum

February 12, 2014 | by

LindseyMy research focuses on a disease known as myelodysplastic syndrome (MDS). MDS is a pre-leukemic bone marrow disorder that is characterized by ineffective production of red blood cells, neutrophils, and/or platelets. MDS occurs either in the elderly or people who have undergone treatment for a previous cancer, such as breast cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma. Currently, the only effective treatment is a bone marrow transplant and patient survival rates are low, with only 35% surviving more than 3 years after diagnosis. The cause of MDS is still unknown and mutations in known cancer-causing genes are rare in patients, so it’s important to find the cause of the disease in order to develop better treatment. In 2011, a paper from Yoshida, et al. was published reporting that the sequencing of bone marrow of 29 patients with MDS revealed mutations in proteins involved in RNA splicing in nearly 50% of patients (1). These mutations were heterozygous and mutually exclusive, with the most common mutations occurring in U2AF1, SRSF2, SF3B1, and ZRSR2. Additional publications from multiple labs have confirmed these results and revealed that these mutations occur early in the disease and remain stable throughout the disease. Researchers think that these mutations could be a possible cause for MDS.

Our lab’s long-term goal is to determine the role of RNA splicing in cancer development, and the publication of the above results provided us with a unique opportunity to study a mutation that may affect RNA splicing and be involved in the early development of MDS. I chose to focus on the SRSF2 gene because it is a well-known protein, and mutations are confined to a single amino acid. SRSF2 is a member of the SR family of proteins that are involved in both constitutive and alternative splicing. In addition, loss of SRSF2 leads to an increase in genome instability, likely due to its role in transcriptional elongation (2, 3). I have developed stable cell lines in bone marrow cells from a patient with erythroleukemia that express either wildtype SRSF2 or SRSF2 with a mutation found in MDS patients. Short term analysis of these cell lines has shown that the mutant cells exhibit both a G2/M cell cycle arrest and increased apoptosis compared to the cells expressing wildtype SRSF2. Cell staining has also shown an increase in γ-H2A.X phosphorylation, an early marker for genome instability, in mutant cells compared to wildtype cells. Whole RNA sequencing has also shown gene expression changes and alternative splicing changes common to all three mutants when compared to the wildtype cell line. Based on our current data, we speculate that SRSF2 amino acid P95 mutations could be a cause of MDS. The primary reason is likely to be more from the genomic instability observed by this mutation rather than from the alternative splicing changes. In fact, recent publications have shown a higher mutation burden in MDS patients with mutations in RNA splicing or DNA methylation pathways than in MDS patients with mutations in transcription, cohesin, or signal transduction pathways (4). In addition, many of the observed splicing changes were changes that often occur under cell stress, such as the alternative splicing of heat shock proteins and cell cycle regulators.

 

1.         Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, Yamamoto R, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 2011;478(7367):64-9. Epub 2011/09/13. doi: 10.1038/nature10496. PubMed PMID: 21909114.

2.         Lin S, Coutinho-Mansfield G, Wang D, Pandit S, Fu X-D. The splicing factor SC35 has an active role in transcriptional elongation. Nature Structural & Molecular Biology. 2008;15(8):819-26. doi: 10.1038/nsmb.1461.

3.         Ji X, Zhou Y, Pandit S, Huang J, Li H, Lin CY, et al. SR proteins collaborate with 7SK and promoter-associated nascent RNA to release paused polymerase. Cell. 2013;153(4):855-68. doi: 10.1016/j.cell.2013.04.028. PubMed PMID: 23663783.

4.         Haferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G, et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, UK. 2013. doi: 10.1038/leu.2013.336. PubMed PMID: 24220272.


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