The Mammalian Cellular Splicing Machine - Structure and Function

描述(申请人提供)：大多数真核细胞前mRNAs含有非编码序列(内含子)，必须去除这些非编码序列(内含子)才能将编码序列(外显子)准确地置于正确的阅读框架中。这一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-剪接体中。虽然对Pre-mRNA剪接的机制已经进行了广泛的研究，但对这一过程的结构和调控仍不是很清楚。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出来。我们从细胞核中分离的复合体比在体外组装的剪接复合体要大得多，因此被称为超剪接小体。我们最近发现，超剪接体由四个活跃的天然剪接体组成，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，可以预期，更好地理解前mRNA剪接的机制应该会导致更好地理解发育和癌症。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：分离的超复制小体代表在剪接反应的不同阶段分离的核前mRNAs的稳定群体。因此，根据转录和剪接阶段开发制备和分离均一超剪接体的方法应该允许我们对结构有更高的分辨率，更好地了解超剪接体中的成分、定位和相互作用，从而更好地了解RNA剪接机的工作原理。具体目标：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，包括利用低温EM单粒子技术进行更高分辨率的结构分析。我们将对在特定转录本上组装的天然剪接体和超剪接体进行分析，包括交替剪接的转录本，并捕获到特定的功能状态(SA#1)。我们还将对这些复合体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被阻止的剪接体组成的差异或与不同的前mRNAs相关的剪接体的组成。将使用标记有金纳米簇(SA#3)的核酸和抗体对超剪接体及其衍生的天然剪接体中的剪接体成分进行定位，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信使核糖核酸重组的超复制小体。这些实验应该有助于识别组装的复合体中的前mRNA途径，并能够定位关键的剪接体组件。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，可以预期，更好地理解前mRNA剪接的机制应该会导致更好地理解发育和癌症。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。

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.