Molecular Analysis of Nuclear Bodies and RNP Trafficking Pathways in the Cell Nucleus

哺乳动物细胞内的许多重要过程被划分在特定的亚细胞结构中。因此，膜结合的细胞质细胞器，如线粒体和溶酶体，分别区室化能量产生和蛋白质降解。核小体（NB），如核仁，提供不被膜包围的专门区室，但仍然浓缩特定的蛋白质和RNA。NB的数量和形态根据细胞类型、细胞生理和生长状态而变化。当细胞对压力或疾病机制（包括病毒感染和癌症）作出反应时，细胞核也经常发生突变，导致遗传性遗传疾病和改变。细胞生长、基因表达和蛋白质产生所需的重要细胞过程，包括mRNA前体转录物的剪接和核糖体亚基的生物合成，都发生在细胞核中。在这些基本过程中所涉及的因素被确定在不同类型的NB的显微镜。例如，核仁是核糖体RNA（rRNA）基因的转录以及随后rRNA的加工和核糖体亚基的组装的位点。大多数哺乳动物基因被转录为大的前体RNA（pre-mRNAs），其必须在细胞核中剪接以去除内含子序列并形成mRNAs。剪接机制包括称为snRNP的RNA-蛋白质亚基和另外的蛋白质剪接因子。这些剪接组分与不同类型的NB相关，包括剪接斑点（染色质间颗粒簇）和Cajal小体（CB），而在核仁中也检测到剪接组分的子集。NB可以在间期和有丝分裂期间组装和拆卸，并且它们的组分分子不断地通过它们运输。因此，通过显微镜检测到的NB的外观代表动态结构的稳态图像。重要的是，NB的大小、形态和分子组成可以响应细胞环境的扰动和变化而迅速改变。尽管核糖体亚基生物合成和前体mRNA剪接过程具有重要的功能重要性和临床意义，但我们仍然缺乏对这些过程如何在细胞核内发生的详细了解，包括rRNA和前mRNA加工机制的组装如何在显微镜检测到的不同NB结构中被划分。分离完整NB的困难意味着它们的分子组成、它们形成的调节以及蛋白质和RNA-蛋白质复合物（RNP）如何在它们之间运输仍然不清楚。该项目旨在提高我们对这些重要结构的机械理解细胞核中的功能关系。我们专注于详细的生物化学分析的NB，使用新的实验方法。我们已经确定了小分子化学工具，我们称之为“NB调节剂”，它可以改变特定NB的结构和组成，包括核仁，斑点和CB。我们将使用这些化学调节剂，结合显微镜和多组学分析，详细说明NB的结构和性质如何受到影响。我们将使用热蛋白质谱鉴定NB调节剂结合靶点。使用高分辨率，基于uHPLC的尺寸排阻色谱，我们将使用基于质谱的蛋白质组学和RNAseq，在纯化的细胞核和纯化的核仁的提取物中分离和纯化核糖体组装复合物和snRNP复合物的组分，所述提取物分离自对照细胞或用不同NB调节剂处理的细胞。我们将分析影响蛋白质和核仁，CB和斑点之间的RNP复合物的运输，方向性和运动速率的机制，结合荧光和电子显微镜的分子测定。

Many important processes within mammalian cells are compartmentalised within specific subcellular structures. Thus, membrane bound cytoplasmic organelles, such as mitochondria and lysosomes, compartmentalise energy production and protein degradation, respectively. Nuclear Bodies (NBs), such as nucleoli, provide specialised compartments that are not surrounded by membranes, but still concentrate specific proteins and RNAs. The number and morphology of NBs varies according to cell type, cell physiology and growth state. NBs are also frequently altered in cells with mutations causing inherited genetic disorders and change when cells respond to stress, or disease mechanisms, including viral infection and cancer.Vital cell processes required for cell growth, gene expression and protein production, including the splicing of mRNA precursor transcripts and the biogenesis of ribosome subunits, take place in the nucleus. The factors involved in these essential processes are identified by microscopy in different types of NBs. For example, the nucleolus is the site of transcription of ribosomal RNA (rRNA) genes and the subsequent processing of rRNA and assembly of ribosome subunits. Most mammalian genes are transcribed as large precursor RNAs (pre-mRNAs), which must be spliced in the nucleus to remove intron sequences and form mRNAs. The splicing machinery comprises RNA-protein subunits, called snRNPs, and additional protein splicing factors. These splicing components associate with different types of NBs, including splicing speckles (clusters of interchromatin granules) and Cajal bodies (CBs), while a subset of splicing components are also detected in the nucleolus.NBs can assemble and disassemble, both in interphase and during mitosis, and their component molecules continually traffic through them. Therefore, the appearance of NBs detected by microscopy represents a steady state image of dynamic structures. Importantly, the size, morphology and molecular composition of NBs can rapidly change in response to perturbations and variation in the cell environment.Despite the major functional importance and clinical relevance of the processes of ribosome subunit biogenesis and pre-mRNA splicing, we still lack a detailed understanding of how these processes take place within the cell nucleus, including how the assembly of both the rRNA and pre-mRNA processing machineries are compartmentalised within the different NB structures that are detected by microscopy. The difficulty in isolating intact NBs means their molecular composition, the regulation of their formation and how proteins and RNA-protein complexes (RNPs) traffic between them, is still not known in detail.This project is designed to improve our mechanistic understanding of these important structure-function relationships in the cell nucleus. We focus on detailed biochemical analyses of NBs, using novel experimental approaches. We have identified small molecule chemical tools, which we term, 'NB modulators', that alter the structure and composition of specific NBs, including nucleoli, speckles and CBs. We will use these chemical modulators, in conjunction with microscopy and poly-omics assays, to characterise in detail how the structures and properties of NBs are affected. We will identify NB modulator binding targets, using thermal protein profiling. Using high resolution, uHPLC-based size exclusion chromatography, we will fractionate and characterise components of ribosome assembly complexes and snRNP complexes, in extracts of purified nuclei and purified nucleoli, isolated from either control cells, or from cells treated with different NB modulators, using both mass spectrometry-based proteomics and RNAseq. We will analyse mechanisms affecting the trafficking, directionality and rate of movement of proteins and RNP complexes between nucleoli, CBs and speckles, combining molecular assays with fluorescence and electron microscopy.