The Mammalian Cellular Splicing Machine - Structure and Function

哺乳动物细胞拼接机 - 结构和功能

• 批准号: 7807999
• 负责人: Ruth Sperling
• 金额: $ 20.85万
• 依托单位: HEBREW UNIVERSITY OF JERUSALEM
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7807999
• 关键词: Accounting; Achievement; Affect; Alternative Splicing; Antibodies; Antisense Oligonucleotides; Arts; Cell Nucleus; Cells; Code; Communication; Complex; Cytoplasm; Defect; Development; Event; Exons; Functional RNA; Gene Expression; Gene Mutation; General Population; Genome; Goals; Gold; Health; Human Pathology; Imagery; In Vitro; Introns; Knowledge; Label; Laboratory Study; Lead; Life; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Messenger RNA; Methodology; Methods; Modification; Nuclear; Nucleic Acids; Nucleoproteins; Pathway interactions; Pentas; Population; Preparation; Process; Protein Splicing; Proteins; Proteomics; Quality Control; RNA; RNA Processing; RNA Splicing; Reaction; Reading Frames; Regulation; Research; Resolution; Ribonucleoproteins; Small Nuclear Ribonucleoproteins; Spliceosomes; Staging; Structure; System; Techniques; Three-Dimensional Image; Time; Transcript; Work; base; comparative; experience; human disease; image reconstruction; in vivo; insight; mRNA Precursor; multicore processor; nanoGold; novel; particle; public health relevance; reconstitution; research study; stable cell line; three dimensional structure

DESCRIPTION (provided by applicant): Most eukaryotic pre-mRNAs contain non-coding sequences (introns) that must be removed in order to accurately place the coding sequences (exons) in the correct reading frame. This critical regulatory event, termed pre-mRNA splicing, is fundamental in development and cancer, and occurs in a multi-component macromolecular machine, the spliceosome. Although the mechanism of pre-mRNA splicing has been extensively studied, the structure and regulation of this process is still not well understood. We have studied the mammalian splicing complex in its intact form, isolated from nuclei of living cells. The complexes we have isolated from cell nuclei are much larger than the splicing complexes assembled in vitro, and are thus termed supraspliceosomes. We have recently shown that the supraspliceosome is composed of four active native spliceosomes, each resembling the in vitro assembled spliceosome, which are connected via the pre-mRNA. Health Relevance: Defects in alternative splicing were correlated with human pathologies and malignancy. It is therefore anticipated that better understanding of the mechanism of pre-mRNA splicing should lead to better understanding of development and cancer. The long term objective of this project is to understand the regulation of splicing and alternative splicing, thus recognizing how defects in these important processes affect human diseases. Hypothesis: The isolated supraspliceosomes represent the steady-state population of nuclear pre-mRNAs that were isolated at different stages of the splicing reaction. It is thus assumed that developing methods for the preparation and isolation of homogeneous supraspliceosomes with respect to their transcript and splicing stage should allow us to get a higher resolution of the structure, a better knowledge of the components, localization and interactions within the supraspliceosome, and thus a better understanding of the working of the RNA splicing machine. Specific Aims: We propose to conduct in-depth structural and functional analyses of the native spliceosome and the supraspliceosome, including higher resolution structural analysis by the cryo-EM single particle techniques. We will perform analyses of native spliceosomes and supraspliceosomes assembled on specific transcripts, including alternatively spliced transcripts, and trapped in specific functional states (SA#1). We will also perform proteomic analyses by mass spectrometry of these complexes (SA#2). This approach might reveal differences in the composition of spliceosomes that were arrested at specific splicing stages or associated with different pre-mRNAs. Localization of spliceosomal components within supraspliceosomes and native spliceosomes derived from them will be performed using nucleic acids and antibodies tagged with gold-nanoclusters (SA#3), followed by cryo-EM structural analyses. The structural analyses will also include supraspliceosomes reconstituted with gold-tagged pre-mRNA. These experiments should assist in identifying the pre-mRNA pathway within the assembled complex and enable the localization of key spliceosomal components. PUBLIC HEALTH RELEVANCE: Defects in alternative splicing were correlated with human pathologies and malignancy. It is therefore anticipated that better understanding of the mechanism of pre-mRNA splicing should lead to better understanding of development and cancer. The long term objective of this project is to understand the regulation of splicing and alternative splicing, thus recognizing how defects in these important processes affect human diseases.

描述（由申请人提供）：大多数真核前mrna包含非编码序列（内含子），必须将其去除，以便将编码序列（外显子）准确地放置在正确的阅读框中。这个关键的调控事件，被称为前mrna剪接，是发育和癌症的基础，发生在一个多组分的大分子机器中，剪接体。虽然pre-mRNA剪接的机制已被广泛研究，但这一过程的结构和调控仍不清楚。我们已经研究了哺乳动物剪接复合体在其完整的形式，从活细胞的细胞核分离。我们从细胞核中分离出的复合体比体外组装的剪接复合体大得多，因此被称为上质体。我们最近表明，超剪接体由四个活性的天然剪接体组成，每个剪接体都类似于体外组装的剪接体，它们通过前mrna连接在一起。健康相关性：选择性剪接缺陷与人类病理和恶性肿瘤相关。因此，预期对pre-mRNA剪接机制的更好理解将导致对发育和癌症的更好理解。该项目的长期目标是了解剪接和选择性剪接的调控，从而认识到这些重要过程中的缺陷如何影响人类疾病。假设：分离的上质体代表在剪接反应的不同阶段分离的核前mrna的稳态群体。因此，我们认为，开发制备和分离同源上质体的方法，就其转录和剪接阶段而言，应该使我们能够获得更高的结构分辨率，更好地了解上质体的成分、定位和相互作用，从而更好地理解RNA剪接机器的工作原理。具体目的：我们建议对天然剪接体和超剪接体进行深入的结构和功能分析，包括使用低温电镜单粒子技术进行更高分辨率的结构分析。我们将分析在特定转录本上组装的天然剪接体和超剪接体，包括选择性剪接转录本，并被困在特定的功能状态（SA#1）。我们还将通过质谱法对这些复合物进行蛋白质组学分析（sa# 2）。这种方法可能揭示剪接体组成的差异，剪接体在特定剪接阶段被阻止或与不同的pre- mrna相关。将使用带有金纳米簇标记的核酸和抗体（SA#3）对剪接体及其衍生的剪接体中的剪接体成分进行定位，然后进行低温电镜结构分析。结构分析还将包括用金标记的pre-mRNA重组的上质体。这些实验应该有助于识别组装复合体内的pre-mRNA通路，并使关键剪接体成分能够定位。公共卫生相关性：选择性剪接缺陷与人类病理和恶性肿瘤相关。因此，预期对pre-mRNA剪接机制的更好理解将导致对发育和癌症的更好理解。该项目的长期目标是了解剪接和选择性剪接的调控，从而认识到这些重要过程中的缺陷如何影响人类疾病。