Transcription and Splicing Dynamics in Single Cells

单细胞中的转录和剪接动力学

• 批准号: 10702544
• 负责人: Daniel Larson
• 金额: $ 249.47万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702544
• 关键词: Acute; Acute Myelocytic Leukemia; Area; Behavior; Biophysics; Blood; Cells; Cellular biology; Clinical; Clonal Hematopoietic Stem Cell; Complement; Computer Analysis; Computer Models; Coupled; Coupling; DNA-Directed RNA Polymerase; Development; Disease; Disease Progression; Dysmyelopoietic Syndromes; Dysplasia; Eukaryotic Cell; Fluorescent Probes; Gene Expression; Gene Expression Alteration; Gene Expression Process; Gene Expression Profiling; Gene Expression Regulation; Genes; Genetic Transcription; Genomic approach; Goals; Health; Hematopoiesis; Heterogeneity; Human; In Vitro; Individual; Ineffective Hematopoiesis; Laboratories; Malignant - descriptor; Marrow; Methods; Microscopy; Modeling; Molecular; Molecular Machines; Mutation; Myeloproliferative disease; Nuclear Structure; Nucleotides; Patients; Play; Population; Process; Prognosis; Proteins; RNA; RNA Splicing; Regulation; Research; Resolution; Ribosomes; Role; Sampling; Site; Spliceosome Assembly Pathway; Spliceosomes; Sum; Time; Tissues; Translations; Variant; Visualization; Work; base; bench to bedside; cytopenia; hematopoietic stem cell differentiation; live cell imaging; macromolecule; peripheral blood; programs; reconstitution; single molecule; stem cells; structural biology; transcriptome sequencing

Gene Expression in Single Cells: Gene expression refers to the sum of processes that enable cells to control their complement of RNA and protein. Megadalton molecular machines such as RNA polymerase, the spliceosome, and the ribosome carry out the synthesis, processing, and translation of RNA. Advances in structural biology have revealed the atomic details of these machines, and the revolution in sequencing has engendered an understanding of their respective enzymatic activities with nucleotide resolution. A central challenge in cell biology is to understand how these processes are coupled and regulated in time and space in single living cells. In recent years, through parallel advances in microscopy, fluorescent probe development, computational modeling, and gene editing it is now possible to directly observe the processes of gene expression (transcription, splicing, translation) in living cells. The Larson lab has played an essential role in this development, for example by being the first lab to visualize transcription and splicing of single human genes in real time. The view that has emerged from these studies is that gene regulation is an extremely dynamic process where the random behavior of individual molecules plays an important role in determining how a cell controls expression. Coupling live-cell imaging with RNA-sequencing based methods is an exciting approach for understanding gene expression in health and disease. Gene Expression in Myeloid Malignancy: The differentiation of hematopoietic stem cells into committed lineages in the blood is the result of concerted regulation between transcription, splicing and translation, and a goal of the laboratory is to understand the dynamic interplay between these processes in single cells. Moreover, mutations in the gene expression machinery drive the development of clonal stem cell disorders termed 'myeloid malignancies.' Specifically, the Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) are a heterogeneous group of malignant clonal hematopoietic stem cell disorders with poor prognosis and few treatment options. MDS is characterized by ineffective hematopoiesis, marrow dysplasia, peripheral blood cytopenia, and a high propensity for transformation into AML, which is an acute proliferative disease. Simply stated, MDS is a disease of differentiation, while AML is a disease of differentiation plus proliferation. Over 60% of MDS patients carry a mutation in the spliceosome, and the Larson lab has proposed a non-canonical role for the splicing machinery in disease progression. Overall, gene regulation in this tissue is exceptionally dependent on post-transcriptional mechanisms, opening new avenues of research for understanding hematopoiesis. Analysis of primary human samples and close coupling with the newly-established Myeloid Malignancies clinical program - "bench-to-bedside-to-bench" - makes this area a vibrant and impactful area of study where basic concepts in gene regulation can be immediately applied to human health. Thus, the projects in the laboratory are loosely divided into several areas: 1) Visualizing spliceosome assembly and splice site selection 2) Deciphering the role of nuclear structure in modulating gene expression 3) Determining the causes and consequences of transcriptional heterogeneity 4) Developing in vitro reconstituted models for examining the coupling between transcription, splicing, translation 5) Computational analysis of gene expression 6) Understanding gene expression alteration in myeloid malignancy.

单个细胞中的基因表达：基因表达是指使细胞能够控制其RNA和蛋白质补体的过程总和。 Megadalton分子机，例如RNA聚合酶，剪接体和核糖体进行RNA的合成，加工和翻译。结构生物学的进步揭示了这些机器的原子细节，并且测序的革命通过核苷酸分辨率使他们对各自的酶促活性有所了解。细胞生物学中的一个核心挑战是了解这些过程如何在单个活细胞中的时间和空间中耦合和调节。近年来，通过显微镜的并行进步，荧光探针开发，计算建模和基因编辑，现在可以直接观察活细胞中基因表达（转录，剪接，翻译）的过程。 Larson Lab在这一发展中发挥了至关重要的作用，例如，是第一个实时可视化转录和剪接的实验室。从这些研究中得出的观点是，基因调节是一个极其动态的过程，在该过程中，单个分子的随机行为在确定细胞如何控制表达方面起着重要作用。与基于RNA的基于基于RNA的方法耦合活细胞成像是一种令人兴奋的方法，用于了解健康和疾病中的基因表达。 髓样恶性肿瘤中的基因表达：将造血干细胞分化为血液中的谱系谱系是由转录，剪接和翻译之间的一致调节的结果，实验室的目标是了解单个细胞中这些过程之间的动态相互作用。此外，基因表达机械中的突变驱动了称为“髓样恶性肿瘤”的克隆干细胞疾病的发展。 具体而言，骨髓增生性综合征（MDS）和急性髓性白血病（AML）是一组异质性的恶性克隆造血干细胞疾病，预后不良，治疗方案很少。 MDS的特征是无效的造血，骨髓发育异常，周围血细胞质症，以及向AML转化的高倾向，这是一种急性增殖疾病。简而言之，MDS是一种分化的疾病，而AML是一种分化和增殖的疾病。超过60％的MDS患者在剪接体中具有突变，Larson Lab提出了疾病进展中剪接机械的非典型作用。总体而言，该组织中的基因调节特别取决于转录后机制，为理解造血的研究开辟了新的研究途径。分析主要人类样品，并与新建立的髓样恶性肿瘤临床计划“基准至床位” - 使该领域成为充满活力和有影响力的研究领域，其中基因调节中的基本概念可以立即应用于人类健康。 Thus, the projects in the laboratory are loosely divided into several areas: 1) Visualizing spliceosome assembly and splice site selection 2) Deciphering the role of nuclear structure in modulating gene expression 3) Determining the causes and consequences of transcriptional heterogeneity 4) Developing in vitro reconstituted models for examining the coupling between transcription, splicing, translation 5) Computational analysis of gene expression 6) Understanding gene expression alteration in髓样恶性肿瘤。