The role of mutant splicing factor SRSF2 in Myelodysplasia

项目摘要 骨髓增生异常（MDS）是一种获得性造血干细胞（HSC）克隆性疾病，在20世纪70年代中期呈上升趋势。 总生存率低的老龄化人群。剪接体关键因子的突变已在 超过50%的患者和SRSF 2中的突变是鉴定的最常见的突变。 SRSF 2突变如何导致MDS尚不清楚。SRSF 2的突变独特地影响脯氨酸， 位置95，在RNA结合结构域的C末端内。我们最近发现，P95基因突变 组氨酸（P95 H）、亮氨酸（P95 L）或精氨酸（P95 R）改变SRSF 2 RRM的结构，导致 改变的RNA结合亲和力和特异性，从而导致异常剪接。 我们的目的是进一步了解SRSF 2突变如何影响体内RNA结合， 剪接改变如何影响造血干细胞的维持和分化。我们特别 寻求1）了解SRSF 2突变如何破坏其体内功能，2）确定途径 在MDS病理学的根源被选择性剪接破坏，和3）开发治疗方法， SRSF 2突变型MDS。 我们将确定RNA靶和RNA靶基序在体内使用RNA免疫沉淀结合 使用UV交联和高通量测序（HITS-CLIP）以及RNAseq来确定 SRSF 2突变影响其体内功能并识别受选择性剪接影响的重要靶点。我们将 确定关键下游靶标的剪接异构体的功能，特别是其他RNA结合的功能 突变SRSF 2选择性结合和剪接的蛋白质和剪接因子。我们将确定 对干细胞维持和造血祖细胞增殖至关重要的可变剪接事件， 分化和确定SRSF 2突变如何改变其阶段和谱系特异性发生。基于 在我们的结构-功能研究中，我们合理地设计了第一代小分子， SRSF 2，我们将进一步开发这些化合物用于SRSF 2突变型MDS的治疗目的。 我们结合了分子和生物学研究、小分子设计和体内方法， 极大地促进了对SRSF 2突变型MDS的理解和治疗。

PROJECT SUMMARY Myelodysplasia (MDS), an acquired clonal disease of the hematopoietic stem cell (HSC), is on the rise in the aging population with poor overall survival. Mutations in key factors of the spliceosome have been identified in over 50% of patients and mutations in SRSF2 are the most frequent mutations identified. How mutations in SRSF2 contribute to MDS is not known. Mutations in SRSF2 uniquely affect proline at position 95, within the C-terminus of the RNA binding domain. We have recently shown that mutations of P95 to histidine (P95H), leucine (P95L), or arginine (P95R) alter the structure of the SRSF2 RRM, resulting in altered RNA binding affinity and specificity, thereby leading to aberrant splicing. Our aim is to further understand how mutations in SRSF2 mutations affect RNA binding in vivo and how altered splicing affects hematopoietic stem cell maintenance and differentiation. Specifically, we seek to 1) understand how mutations in SRSF2 disrupt its function in vivo, 2) determine the pathways disrupted by alternative splicing at the root of MDS pathology, and 3) develop therapeutic approaches for SRSF2 mutant MDS. We will determine RNA targets and RNA target motifs in vivo using RNA immunoprecipitation in conjunction with UV crosslinking and high throughput sequencing (HITS-CLIP) as well as RNAseq to determine how SRSF2 mutations affect its function in vivo and identify essential targets affected by alternative splicing. We will determine the function of splice isoforms of critical down-stream targets, in particular of other RNA binding proteins and splicing factors that are alternatively bound and spliced by mutant SRSF2. We will identify alternative splice events critical to stem cell maintenance and hematopoietic progenitor proliferation and differentiation and determine how SRSF2 mutations alter their stage and lineage specific occurrence. Based on our structure-function studies we have rationally designed first-generation small molecules that target SRSF2, and we will further develop these compounds for therapeutic purposes of SRSF2 mutant MDS. Our combined molecular and biologic studies, small molecule design, and in vivo approaches promise to greatly advance understanding and treatment of SRSF2 mutant MDS.