Crosstalk of Splicing and Signaling in HSPC fate choices

HSPC 命运选择中剪接和信号传导的串扰

• 批准号: 10434965
• 负责人: Teresa V Bowman
• 金额: $ 40.59万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10434965
• 关键词: Affect; Blood; Cell Culture Techniques; Cells; Chemical Modifier; Chemicals; Coupled; Critical Pathways; Data; Defect; Disease; Dysmyelopoietic Syndromes; Functional disorder; Genetic Screening; Glucocorticoid Receptor; Glucocorticoids; Hematopoietic stem cells; Heterozygote; Human; Human Cell Line; Ineffective Hematopoiesis; Lead; Malignant Neoplasms; Mediating; Modeling; Molecular; Mutate; Mutation; Outcome; Pathway interactions; Patients; Persons; Pharmaceutical Preparations; Pilot Projects; Process; Protein Isoforms; Public Health; Publishing; RNA Splicing; Receptor Signaling; Regulation; Reporter; Reporting; Research; Role; STAT3 gene; Shapes; Siblings; Signal Pathway; Signal Transduction; Specificity; Stat3 protein; Stem Cell Development; Testing; Tissues; Western Blotting; Yeasts; Zebrafish; base; cytopenia; genetic approach; in vivo; insight; loss of function; mRNA Precursor; mutant; novel; novel strategies; novel therapeutic intervention; overexpression; transcriptome sequencing

Crosstalk of Splicing and Signaling during HSPC Fate Choices Project Abstract Myelodysplastic syndrome (MDS) is a spectrum of disorders arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis and cytopenias. Spliceosomal components are mutated in greater than 60% of all MDS cases, yet it remains a puzzle how dysfunction in general splicing factors can mediate the specific outcomes observed in MDS. The slow progress comes, in part, due to our poor understanding of splicing regulation of HSPC fate choices. Indeed, as RNA splicing is mostly studied in yeast and cell culture, tissue-specific regulation of splicing in vivo is largely unexplored. Here, using HSPC development as a paradigm, we seek to illuminate how the molecular regulation of RNA splicing impacts HSPC fate choices. By understanding this fundamental process, we strive to identify potentially novel ways to target splicing factor-defective HSPCs in MDS. We recently reported a severe HSPC formation defect in zebrafish loss-of-function mutants for the spliceosomal component, splicing factor 3b, subunit 1 (sf3b1), which is the most commonly mutated splicing factor in MDS. We uncovered that Sf3b1 regulates stat3 (signal transducer and activator of transcription 3) pre-mRNA splicing and demonstrated that mis-splicing of stat3 results in diminished pathway activation. We determined that overexpression of an active form of Stat3 can suppress the HSPC defects in sf3b1 mutants, demonstrating that Sf3b1 regulation of STAT3 signaling is important for HSPC formation. In preliminary studies, we determined that STAT3 inhibition serves as a conserved synthetic lethality with SF3B1 heterozygosity in both zebrafish and human MDS HSPCs. Based on these findings, we performed a chemical modifier screen in sf3b1 mutant zebrafish and identified other Sf3b1- regulated signaling pathways critical for HSPCs. These studies establish zebrafish as an excellent model for discovery of HSPC splicing regulation and novel MDS vulnerabilities. Here, we will test the hypothesis that the splicing factor Sf3b1 is a key director of the choice of splice isoforms for signaling pathway components essential for HSPC formation. We will use zebrafish genetic and screening capabilities coupled with studies in human MDS cells to explore the role of specific splice isoforms on HSPC fate decisions, determine the function of cis-regulatory sequences on Sf3b1-regulated splicing choices in vivo, and identify novel vulnerabilities for SF3B1-mutated HSPCs. Accomplishing these aims will inform our basic understanding of the poorly understood role of splicing in HSPC fate decisions and lead to the identification of novel approaches for selective targeting of SF3B1-mutated MDS HSPCs.

HSPC 命运选择过程中剪接和信号传导的串扰 项目摘要 骨髓增生异常综合征（MDS）是由造血干细胞和祖细胞引起的一系列疾病 细胞（HSPC）功能障碍导致无效造血和血细胞减少。剪接体成分是 超过 60% 的 MDS 病例发生突变，但一般剪接功能障碍如何发生仍然是一个谜 因素可以介导 MDS 中观察到的特定结果。进展缓慢的部分原因是我们 对 HSPC 命运选择的剪接调控了解甚少。事实上，RNA 剪接的研究主要集中在 在酵母和细胞培养中，体内剪接的组织特异性调节在很大程度上尚未被探索。这里，使用 HSPC 以开发为范式，我们试图阐明 RNA 剪接的分子调控如何影响 HSPC 的命运选择。通过了解这一基本过程，我们努力寻找潜在的新颖方法 靶向 MDS 中剪接因​​子缺陷的 HSPC。我们最近报告了严重的 HSPC 形成缺陷 斑马鱼剪接体成分、剪接因子 3b、亚基 1 (sf3b1) 的功能丧失突变体， 是 MDS 中最常见的突变剪接因子。我们发现 Sf3b1 调节 stat3（信号 转导子和转录激活子 3) pre-mRNA 剪接并证明 stat3 的错误剪接 导致通路激活减少。我们确定 Stat3 活性形式的过度表达可以 抑制 sf3b1 突变体中的 HSPC 缺陷，证明 Sf3b1 对 STAT3 信号传导的调节 对于 HSPC 的形成很重要。在初步研究中，我们确定 STAT3 抑制可作为 在斑马鱼和人类 MDS HSPC 中具有 SF3B1 杂合性的保守合成致死率。基于 根据这些发现，我们在 sf3b1 突变斑马鱼中进行了化学修饰剂筛选，并鉴定了其他 Sf3b1- 对 HSPC 至关重要的调控信号通路。这些研究将斑马鱼确立为一个优秀的模型 HSPC 剪接调节和新的 MDS 漏洞的发现。在这里，我们将检验以下假设： 剪接因子 Sf3b1 是信号通路成分剪接同工型选择的关键指导者 对于 HSPC 的形成至关重要。我们将利用斑马鱼的遗传和筛选能力以及以下方面的研究： 人类MDS细胞探索特定剪接亚型对HSPC命运决定的作用，确定功能 体内 Sf3b1 调节剪接选择的顺式调节序列，并识别新的漏洞 SF3B1 突变的 HSPC。实现这些目标将使我们对贫困人口有基本的了解 了解剪接在 HSPC 命运决定中的作用，并确定了剪接的新方法 选择性靶向 SF3B1 突变的 MDS HSPC。