RNA Processing Machines in Biology and Disease

项目摘要/摘要 这项工作的目标是了解人类剪接体的致病作用和基本生物学。 前信使核糖核酸的剪接是剪接体最广为人知的功能，但这一机制也包含RNA/DNA 结合在基因表达的许多步骤中起作用的蛋白质。该实验室专注于运动神经元病。 肌萎缩侧索硬化症(ALS)和血癌。超过三分之一的肌萎缩侧索硬化症致病基因编码 RNA/DNA结合蛋白，但其功能尚不清楚。我们的新研究导致了令人兴奋的 发现其中三种蛋白质(FUS、TAF15、MATR3)对一组抗原的表达是必不可少的 呈递基因在免疫系统中起着关键的保护作用。值得注意的是，这三个ALS基因 也是表达抗原提呈基因的主转录控制因子所必需的。在……里面 肌萎缩侧索硬化症，免疫系统的过度激活是众所周知的它对运动神经元的有害影响。然而， 免疫系统在ALS中正成为一把双刃剑，研究表明，免疫系统失去了保护作用 功能也会导致运动神经元死亡。目前，人们对这种保护作用知之甚少。至 确定突变的ALS基因导致的抗原提呈因子丢失是否与人类ALS有关 胚胎干细胞将经过CRISPR编辑，以在RNA/DNA结合中包含导致ALS的突变 基因。胚胎干细胞将分化为小胶质细胞，小胶质细胞是中枢神经的免疫细胞。 系统。共培养系统将被用来确定突变的小胶质细胞如何影响运动神经元。 蛋白质组学、转录组学和功能分析将用于评估对小胶质细胞和运动的影响。 神经元。这项工作的一个关键目标是确定抗原提呈因子的丢失是否可以 延伸到其他形式的肌萎缩侧索硬化。如果是这样的话，针对这些因素的疗法可能会令人兴奋。 对多种类型的肌萎缩侧索硬化症有效。我们的另一个研究项目的目标是确定 剪接体蛋白SF3B1与血癌有关。我们CRISPR编辑的ES细胞含有SF3B1 癌症突变，并将使ES细胞分化为造血系。转录组学/蛋白质组学和 分化的影响将被用来确定可能导致SF3B1癌症的变化。我们确认了 一组错误拼接不同SF3B1癌症常见的签名基因，并将确定它们如何错误拼接。 剪接会影响分化。导致SF3B1癌症的候选签名的错误拼接 将使用反义技术进行纠正，目标是将该技术开发为治疗性技术。最后， 我们发现了两个仅在剪接体突变的骨髓增生异常综合征中显著上调的基因。 (MDS)，这是与剪接体突变相关的主要血癌类型。两个上调的基因都发挥了作用 在造血中的关键作用。他们对MDS的潜在贡献将通过造血学来检验 分化试验。总而言之，这项研究将导致在识别缺陷的角色方面取得重要进展 在肌萎缩侧索硬化症和血液病癌症中剪接小体基因，以及帮助确定新的治疗靶点。

PROJECT SUMMARY / ABSTRACT The goal of this work is to understand the disease-causative roles and basic biology of the human spliceosome. Pre-mRNA splicing is the best-known function of the spliceosome, but this machinery also houses RNA/DNA binding proteins with roles in many steps of gene expression. The lab focuses on the motor neuron disease amyotrophic lateral sclerosis (ALS) and on blood cancers. Greater than one third of ALS-causative genes encode RNA/DNA binding proteins yet their functions are not well understood. Our new research led to the exciting discovery that three of these proteins (FUS, TAF15, MATR3) are essential for expression of a set of antigen presentation genes, which play critical protective roles in the immune system. Remarkably, the three ALS genes are also required for expression of the master transcription control factor of the antigen presentation genes. In ALS, hyperactivation of the immune system is known for its detrimental effects on motor neurons. However, the immune system is emerging as a double-edged sword in ALS as studies indicate that loss of its protective functions also contributes to motor neuron death. At present, little is known about the protective roles. To determine whether loss of the antigen presentation factors due to mutant ALS genes contributes to ALS, human embryonic stem (ES) cells will be CRISPR-edited to harbor ALS-causative mutations in the RNA/DNA binding genes. The ES cells will be differentiated into microglia, which are the immune cells of the central nervous system. Co-culture systems will be used to determine how motor neurons are affected by the mutant microglia. Proteomics, transcriptomics, and functional assays will be used to assess effects on microglia and motor neurons. A critical objective of the work is to determine whether loss of the antigen presentation factors can be extended to other forms of ALS. If so, it raises the exciting possibility that therapies targeting these factors may be efficacious for multiple types of ALS. The goal of our other research project is to determine how mutation of the spliceosomal protein SF3B1 contributes to blood cancers. We CRISPR-edited ES cells to harbor an SF3B1 cancer mutation and will differentiate the ES cells into hematopoietic lineages. Transcriptomics/proteomics and impacts on differentiation will be used to identify changes that may contribute to SF3B1 cancers. We identified a set of mis-splicing signature genes common to different SF3B1 cancers and will determine how their mis- splicing affects differentiation. Mis-splicing of signatures that are candidates for contributing to SF3B1 cancers will be corrected using antisense technology, with the goal of developing the technology as a therapeutic. Finally, we identified two genes that are robustly upregulated only in spliceosome-mutated myelodysplastic syndrome (MDS), which is main type of blood cancer associated with spliceosome mutations. Both upregulated genes play key roles in hematopoiesis. Their potential contribution to MDS will be examined using the hematopoietic differentiation assays. Together, this research will lead to important advances in identifying the roles of defective spliceosomal genes in both ALS and hematological cancers as well as aid in identifying new therapeutic targets.