RNA Processing Machines in Biology and Disease

项目总结/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。 前体mRNA剪接是剪接体最著名的功能，但这种机制也容纳RNA/DNA 在基因表达的许多步骤中起作用的结合蛋白。该实验室专注于运动神经元疾病 肌萎缩侧索硬化症（ALS）和血癌。超过三分之一的ALS致病基因编码 RNA/DNA结合蛋白，但它们的功能还不清楚。我们的新研究带来了令人兴奋的 发现这些蛋白质中的三种（FUS、TAF 15、MATR 3）对于一组抗原的表达是必需的 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， 也是抗原呈递基因的主转录控制因子表达所必需的。在 ALS，免疫系统的过度激活以其对运动神经元的有害影响而闻名。但 免疫系统在ALS中是一把双刃剑，因为研究表明， 功能也导致运动神经元死亡。目前，对保护作用知之甚少。到 确定由于突变型ALS基因引起的抗原呈递因子的丧失是否有助于ALS、人ALS和人ALS。 胚胎干（ES）细胞将被CRISPR编辑，以在RNA/DNA结合中携带ALS致病突变， 基因. ES细胞将分化为小胶质细胞，这是中枢神经的免疫细胞 系统共培养系统将用于确定运动神经元如何受到突变小胶质细胞的影响。 蛋白质组学、转录组学和功能测定将用于评估对小胶质细胞和运动神经元的影响。 神经元这项工作的一个关键目标是确定抗原呈递因子的丢失是否可以被 扩展到其他形式的ALS。如果是这样的话，它提出了一种令人兴奋的可能性，即针对这些因素的治疗可能 对多种类型的ALS有效。我们另一个研究项目的目标是确定 剪接体蛋白SF 3B 1与血癌有关。我们用CRISPR编辑ES细胞， 癌症突变，并将ES细胞分化为造血谱系。转录组学/蛋白质组学和 对分化的影响将用于识别可能导致SF 3B 1癌症的变化。我们确定 一组在不同SF 3B 1癌症中常见的错误剪接标记基因，并将决定他们的错误剪接是如何发生的。 剪接影响分化。导致SF 3B 1癌症的候选标记的错误剪接 将使用反义技术进行纠正，目标是将该技术开发为治疗方法。最后， 我们鉴定了两个基因，它们仅在剪接体突变的骨髓增生异常综合征中显著上调， (MDS)是剪接体突变相关的主要血癌类型。这两种上调的基因 造血的关键作用。他们对MDS的潜在贡献将使用造血功能检查。 分化测定。总之，这项研究将导致在确定有缺陷的人的角色方面取得重要进展。 在ALS和血液癌症中的剪接体基因，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.