Crosstalk of Splicing and Signaling in HSPC fate choices

HSPC命运选择过程中剪接和信令的串扰 项目摘要 骨髓增生异常综合征(MDS)是一种由造血干和祖细胞引起的疾病 细胞(HSPC)功能障碍导致无效的造血和细胞减少。剪接体的成分是 在所有MDS病例中，超过60%的病例发生突变，但普遍剪接功能障碍如何发生仍是一个谜 在MDS观察到的特定结果中，因素可以起中介作用。进展缓慢，部分原因是我们的 对剪接调控HSPC命运选择认识不深。事实上，由于RNA剪接主要是在 在酵母和细胞培养中，对体内剪接的组织特异性调控在很大程度上还没有被探索。在这里，使用HSPC 作为一种范式，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方法来 靶剪接因子-MDS中有缺陷的HSPC。我们最近报道了一例严重的HSPC形成缺陷。 斑马鱼剪接体组分剪接因子3b亚基1(SF3B1)功能缺失突变体 是MDS中最常见的突变剪接因子。我们发现SF3B1调节STAT3(信号 转录转导和激活因子3)前mRNA剪接，并证明STAT3的错误剪接 导致通路激活减弱。我们确定过表达一种活性形式的STAT3可以 抑制SF3B1突变体中的HSPC缺陷，表明SF3B1对STAT3信号的调节 对HSPC的形成很重要。在初步研究中，我们确定STAT3抑制作为一种 斑马鱼和人MDS HSPC中具有SF3B1杂合性的保守合成致死性。基于 这些发现，我们在SF3B1突变斑马鱼中进行了化学修饰筛选，并鉴定了其他SF3B1- 受调控的信号通路对HSPC至关重要。这些研究确立了斑马鱼是一种优秀的 发现HSPC剪接调控和新的MDS漏洞。在这里，我们将检验这一假设 剪接因子SF3B1是选择信号通路组件剪接异构体的关键因子 对于HSPC的形成是必不可少的。我们将利用斑马鱼的遗传和筛查能力，结合 探讨人MDS细胞特异性剪接异构体对HSPC命运的决定作用，确定其功能 体内SF3B1调控的剪接选择上的顺式调控序列，并识别新的脆弱性 SF3B1突变的HSPC。实现这些目标将使我们对穷人的基本认识有所了解 了解剪接在HSPC命运决定中的作用，并导致确定新的方法 选择性靶向SF3B1突变的MDS HSPC。

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.