RNA Processing Machines in Biology and Disease

生物学和疾病中的 RNA 加工机器

• 批准号: 10406443
• 负责人: DANESH MOAZED
• 金额: $ 66.39万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10406443
• 关键词: Affect; Amyotrophic Lateral Sclerosis; Antigen Presentation; Antisense Technology; Biological Assay; Biology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; DNA Binding; DNA-Binding Proteins; Defect; Disease; Dysmyelopoietic Syndromes; Gene Expression; Genes; Goals; Hematologic Neoplasms; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Human Biology; Immune; Immune system; Malignant Neoplasms; Microglia; Motor Neuron Disease; Motor Neurons; Mutate; Mutation; Neuraxis; Pathogenesis; Play; Proteins; Proteomics; RNA; RNA Processing; RNA Splicing; RNA, Messenger, Splicing; Research; Research Project Grants; Role; Spliceosomes; System; TAF15 gene; Technology; Therapeutic; Transcriptional Regulation; Work; causal variant; embryonic stem cell; genetic signature; hematopoietic differentiation; human disease; human embryonic stem cell; mRNA Precursor; mutant; neuron loss; new therapeutic target; targeted treatment; transcriptomics

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.

项目摘要 /摘要 这项工作的目的是了解人类剪接体的疾病作用和基本生物学。 前MRNA剪接是剪接体的最著名功能，但该机械也容纳RNA/DNA 结合蛋白质在基因表达的许多步骤中具有作用。实验室重点是运动神经元疾病 肌萎缩性侧索硬化症（ALS）和血液癌。超过三分之一的ALS促疾病基因编码 RNA/DNA结合蛋白的功能尚不清楚。我们的新研究导致了令人兴奋的 发现这些蛋白质中的三种（FUS，TAF15，MATR3）对于表达一组抗原至关重要 表现基因，在免疫系统中起关键的保护作用。值得注意的是，三个ALS基因 也需要表达抗原表现基因的主转录控制因子。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。但是， 由于研究表明其保护性丧失 功能还会导致运动神经元死亡。目前，对保护作用知之甚少。到 确定因突变ALS基因引起的抗原表现因子的丧失是否有助于ALS，人类 将对胚胎干细胞（ES）细胞进行crispr编辑，以携带RNA/DNA结合中的Als aussative突变 基因。 ES细胞将分为小胶质细胞，这些小胶质细胞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的影响 神经元。这项工作的关键目的是确定抗原表现因子的损失是否可以是 扩展到其他形式的ALS。如果是这样，它会增加针对这些因素的治疗的令人兴奋的可能性 对于多种类型的ALS有效。我们另一个研究项目的目标是确定如何突变 剪接体蛋白SF3B1有助于血液癌。我们将ES细胞列出了SF3B1 癌症突变，并将ES细胞区分为造血谱系。转录组学/蛋白质组学和 对差异化的影响将用于确定可能导致SF3B1癌症的变化。我们确定了 不同SF3B1癌症常见的一组错误切开的签名基因，将决定它们的错误 剪接会影响分化。签名的错误拆开，这些签名是为SF3B1癌症贡献的候选者 将使用反义技术纠正，以开发该技术作为治疗性。最后， 我们鉴定了两个仅在剪接体突变的骨髓增生综合征中牢固上调的基因 （MDS），这是与剪接体突变相关的血液癌的主要类型。两个上调的基因发挥 造血作用的关键作用。他们对MD的潜在贡献将使用造血性检查 分化测定。这项研究将导致确定有缺陷的作用的重要进展 ALS和血液癌中的剪接体基因，并有助于鉴定新的治疗靶标。