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: 7621238
• 负责人: Ruth Sperling
• 金额: $ 21.06万
• 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/7621238
• 关键词: Accounting; Achievement; Affect; Alternative Splicing; Antibodies; Antisense Oligonucleotides; Arts; Cell Nucleus; Cells; Code; Communication; Complex; Cytoplasm; Defect; Depth; Development; Event; Exons; Functional RNA; Gene Expression; Gene Mutation; General Population; Genome; Gold; Health; Heterogeneous Nuclear RNA; 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; Numbers; Pathway interactions; Pentas; Population; Preparation; Process; Protein Splicing; Proteins; Proteomics; Public Health; Quality Control; RNA; RNA Processing; RNA Splicing; Reaction; Reading Frames; Regulation; Research; Resolution; Ribonucleoproteins; Small Nuclear Ribonucleoproteins; Spliceosomes; Staging; Structure; System; Techniques; Three-Dimensional Imaging; Time; Transcript; Work; base; comparative; experience; human disease; in vivo; insight; mRNA Precursor; multicore processor; nanoGold; novel; particle; reconstitution; reconstruction; research study; stable cell line

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）：大多数真核前体mRNA含有非编码序列（内含子），为了将编码序列（外显子）准确放置在正确的阅读框中，必须将其去除。这种关键的调节事件，称为前mRNA剪接，是发育和癌症的基础，并发生在多组分大分子机器剪接体中。尽管前体mRNA剪接的机制已经被广泛研究，但这一过程的结构和调控仍然没有很好地理解。我们已经研究了哺乳动物剪接复合体的完整形式，从活细胞的细胞核中分离。我们从细胞核中分离的复合物比体外组装的剪接复合物大得多，因此被称为超剪接体。我们最近发现，superpliceosome是由四个活跃的天然spliceosome，每个类似于在体外组装的spliceosome，这是通过前mRNA连接。健康相关性：选择性剪接缺陷与人类病理学和恶性肿瘤相关。因此，预计更好地了解前mRNA剪接的机制将有助于更好地了解发育和癌症。该项目的长期目标是了解剪接和选择性剪接的调节，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：分离的超剪接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，它是假设，开发方法的制备和分离的同质supraspliceosomes相对于他们的转录和剪接阶段，应该让我们得到更高的分辨率的结构，更好地了解的组成部分，定位和相互作用的supraspliceosome，从而更好地了解RNA剪接机的工作。具体目标：我们建议进行深入的结构和功能分析的本地spliceosome和supraspliceosome，包括更高分辨率的结构分析的冷冻EM单粒子技术。我们将对在特定转录物上组装的天然剪接体和超剪接体进行分析，包括选择性剪接转录物，并被困在特定的功能状态中（SA#1）。我们还将通过这些复合物的质谱法进行蛋白质组学分析（SA#2）。这种方法可能揭示在特定剪接阶段或与不同前mRNA相关的剪接体组成的差异。将使用用金纳米簇（SA#3）标记的核酸和抗体进行剪接体组分在超剪接体和源自它们的天然剪接体内的定位，然后进行冷冻-EM结构分析。结构分析还将包括用金标记的前mRNA重建的超剪接体。这些实验应有助于确定组装复合物内的前mRNA通路，并使关键剪接体组分的定位成为可能。公共卫生相关性：可变剪接缺陷与人类病理学和恶性肿瘤相关。因此，预计更好地了解前mRNA剪接的机制将有助于更好地了解发育和癌症。该项目的长期目标是了解剪接和选择性剪接的调节，从而认识到这些重要过程中的缺陷如何影响人类疾病。