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）：大多数真核生物前MRNA包含非编码序列（内含子），必须将其删除才能将编码序列（外显子）精确放置在正确的阅读框中。这个关键的调节事件称为前MRNA剪接，在发育和癌症中是基础，并且发生在多组分大分子机器中，即剪接体。尽管已经对MRNA剪接的机制进行了广泛的研究，但该过程的结构和调节仍未得到充分了解。我们已经研究了从活细胞核分离的完整形式的哺乳动物剪接复合物。我们从细胞核中分离出的络合物比在体外组装的剪接复合物大得多，因此称为上斜肌小体。我们最近表明，上斜肌组由四个活跃的天然剪接体组成，每个剪接体类似于体外组装的剪接体，这些剪接体通过前MRNA连接。健康相关性：替代剪接中的缺陷与人类病理和恶性肿瘤相关。因此，预计更好地了解MRNA前剪接机理应该会更好地理解发展和癌症。该项目的长期目标是了解剪接和替代剪接的调节，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：孤立的上斜长皮小体代表核前体内的稳态种群，在剪接反应的不同阶段分离。因此，假定开发用于制备和隔离同质性上椎板的方法相对于其转录和剪接阶段的方法，应使我们能够获得更高的结构分辨率，更好地了解组件的了解，对Supraspliceoss中的本地化和相互作用，从而更好地了解RNA拼接机的工作。具体目的：我们建议对天然剪接体和上斜杆面的深入结构和功能分析进行深入的结构和功能分析，包括通过冷冻EM单个粒子技术进行更高分辨率的结构分析。我们将对在特定的转录本上组装的天然剪接体和上置质体进行分析，包括剪接的转录本，并将其捕获在特定的功能状态（SA＃1）中。我们还将通过这些复合物的质谱法（SA＃2）进行蛋白质组学分析。这种方法可能揭示了在特定剪接阶段停滞或与不同的MRNA相关的剪接体组成的差异。将使用核酸和用金纳米群（SA＃3）标记的核酸和抗体进行剪接体成分的定位和源自其中的天然剪接体的定位，然后进行冷冻EM结构分析。结构分析还将包括与金标记的前MRNA重构的上斜肌体。这些实验应有助于识别组装络合物内的MRNA途径，并使钥匙夹层成分的定位。公共卫生相关性：替代剪接的缺陷与人类病理和恶性肿瘤有关。因此，预计更好地了解MRNA前剪接机理应该会更好地理解发展和癌症。该项目的长期目标是了解剪接和替代剪接的调节，从而认识到这些重要过程中的缺陷如何影响人类疾病。