The role of mutant splicing factor SRSF2 in Myelodysplasia

突变剪接因子SRSF2在骨髓增生异常中的作用

• 批准号: 9312797
• 负责人: Stephanie Halene
• 金额: $ 37.16万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-15 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9312797
• 关键词: Affect; Affinity; Alternative Splicing; Amino Acids; Arginine; Binding; Biological Assay; Cell Differentiation process; Cell Maintenance; Cell model; Cells; Characteristics; Clonal Expansion; Data; Databases; Development; Disease; Dysmyelopoietic Syndromes; Event; Functional disorder; Gene Expression; Generations; Genes; Genome; Goals; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Heterogeneous-Nuclear Ribonucleoproteins; High-Throughput Nucleotide Sequencing; Histidine; Immunoprecipitation; In Vitro; Lead; Leucine; Maps; Messenger RNA; Modeling; Molecular; Mus; Mutation; Nature; Pancytopenia; Pathology; Pathway interactions; Patients; Pattern; Phenotype; Plant Roots; Population; Positioning Attribute; Proline; Protein Isoforms; Protein Splicing; Proteins; RNA; RNA Binding; RNA Processing; RNA Recognition Motif; RNA Splicing; RNA-Binding Proteins; Recurrence; Regulation; Role; SRSF2 gene; Specificity; Spliced Genes; Spliceosomes; Stem cells; Structure; Technology; Testing; Therapeutic; Transcriptional Regulation; Xenograft procedure; actionable mutation; aging population; base; crosslink; crosslinking and immunoprecipitation sequencing; design; disease-causing mutation; epigenome; in vivo; inhibitor/antagonist; insight; mimicry; mutant; novel; peripheral blood; progenitor; programs; protein expression; protein function; small molecule; stem; tool; transcriptome; transcriptome sequencing

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.

项目总结 骨髓发育不良(MDS)是一种获得性造血干细胞(HSC)克隆性疾病，近年来其发病率呈上升趋势。 人口老龄化，总体生存状况不佳。剪接体关键因子的突变已在 超过50%的患者和SRSF2突变是最常见的突变。 SRSF2的突变如何导致MDS尚不清楚。SRSF2基因突变独一无二地影响 第95位，位于RNA结合域的C-末端。我们最近发现，P95的突变 组氨酸(P95H)、亮氨酸(P95L)或精氨酸(P95R)改变SRSF2 RRM的结构，导致 改变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SRSF2突变如何影响体内RNA结合和 改变的剪接如何影响造血干细胞的维持和分化。具体来说，我们 试图1)了解SRSF2的突变是如何在体内破坏其功能的，2)确定途径 在MDS病理的根源上被选择性剪接破坏，以及3)开发治疗方法 SRSF2突变体MDS。 我们将联合使用RNA免疫沉淀在体内确定RNA靶标和RNA靶标基序 通过UV交联和高通量测序(HITS-CLIP)以及RNAseq来确定如何 SRSF2突变在体内影响其功能，并识别受选择性剪接影响的重要靶点。我们会 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 由突变体SRSF2交替结合和剪接的蛋白质和剪接因子。我们将确定 选择性剪接事件对干细胞维持和造血祖细胞增殖至关重要 并确定SRSF2突变如何改变其阶段和谱系特定的发生。基座 在我们的结构-功能研究中，我们合理地设计了第一代靶向小分子 SRSF2，我们将进一步开发这些化合物用于SRSF2突变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，前景看好 以促进对SRSF2突变型MDS的认识和治疗。