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: 9973436
• 负责人: Teresa V Bowman
• 金额: $ 41.5万
• 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/9973436
• 关键词: Affect; Blood; Cell Culture Techniques; Cells; Chemical Modifier; Chemicals; Coupled; Critical Pathways; Data; Defect; Development; 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; 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命运选择过程中的剪接和信号串扰 项目摘要 骨髓增生异常综合征（MDS）是一系列造血干细胞和祖细胞异常的疾病， 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 在超过60%的MDS病例中发生了突变，但仍有一个难题， 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， 对HSPC命运选择的剪接调控了解不足。事实上，由于RNA剪接的研究主要集中在 酵母和细胞培养物中，体内剪接的组织特异性调节在很大程度上未被探索。在这里，使用HSPC 作为一个范例，我们试图阐明RNA剪接的分子调控如何影响 HSPC命运选择。通过了解这一基本过程，我们努力确定潜在的新方法， 靶向MDS中剪接因子缺陷的HSPC。我们最近报道了一个严重的HSPC形成缺陷， 斑马鱼剪接体组分剪接因子3b亚基1（sf 3b 1）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Sf 3b 1调节stat 3（信号 3）前体mRNA剪接，并证明stat 3的错误剪接 导致减少的途径激活。我们确定过表达活化形式的Stat 3可以 抑制sf 3b 1突变体中的HSPC缺陷，表明Sf 3b 1对STAT 3信号传导的调节是 对HSPC的形成很重要。在初步研究中，我们确定STAT 3抑制作为一种免疫抑制剂， 在斑马鱼和人MDS HSPC中具有SF 3B 1杂合性的保守合成致死性。基于 根据这些发现，我们在sf 3b 1突变斑马鱼中进行了化学修饰剂筛选，并鉴定了其他Sf 3b 1- 调控HSPC的关键信号通路。这些研究使斑马鱼成为一种极好的模型， 发现HSPC剪接调控和新的MDS漏洞。在这里，我们将测试假设， 剪接因子Sf 3b 1是选择信号通路组分剪接异构体的关键指导者 对HSPC形成至关重要。我们将利用斑马鱼的遗传和筛选能力， 人MDS细胞，以探索特定剪接异构体对HSPC命运决定的作用，确定其功能， 顺式调控序列对Sf 3b 1调节的剪接选择在体内，并确定新的脆弱性， SF 3B 1突变的HSPC。实现这些目标将使我们对穷人的基本认识更加深刻。 了解剪接在HSPC命运决定中的作用，并导致识别新的方法， 选择性靶向SF 3B 1突变的MDS HSPC。