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为特定的亚细胞结构。因此，膜结合细胞器，如线粒体和溶酶体，分别负责能量的产生和蛋白质的降解。核体（NBs），如核仁，提供不被膜包围的特殊隔室，但仍然集中特定的蛋白质和rna。NBs的数量和形态随细胞类型、细胞生理和生长状态的不同而不同。NBs在细胞中也经常发生改变，发生突变导致遗传疾病，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，NBs也会发生变化。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发生，都发生在细胞核中。参与这些基本过程的因素是通过显微镜在不同类型的NBs中确定的。例如，核仁是核糖体RNA （rRNA）基因转录和随后的rRNA加工和核糖体亚基组装的位点。大多数哺乳动物基因转录为大前体rna (pre- mrna)，必须在细胞核中剪接以去除内含子序列并形成mrna。剪接机制包括rna -蛋白质亚基（称为snRNPs）和额外的蛋白质剪接因子。这些剪接成分与不同类型的NBs相关，包括剪接斑点（染色质间颗粒簇）和Cajal体（CBs），而在核仁中也检测到剪接成分的子集。NBs可以在间期和有丝分裂过程中组装和拆卸，它们的组成分子不断地通过它们进行运输。因此，显微镜检测到的NBs的外观代表了动态结构的稳态图像。重要的是，NBs的大小、形态和分子组成可以随着细胞环境的扰动和变化而迅速改变。尽管核糖体亚基生物发生和前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下检测到的不同NB结构中被划分。分离完整的NBs的困难意味着它们的分子组成、它们的形成调控以及蛋白质和rna -蛋白复合物（RNPs）如何在它们之间传输，仍然是未知的。该项目旨在提高我们对细胞核中这些重要结构-功能关系的机制理解。我们专注于NBs的详细生化分析，使用新颖的实验方法。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，包括核核、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，详细描述NBs的结构和性质是如何受到影响的。我们将确定NB调节剂结合目标，使用热蛋白分析。使用高分辨率，uhplc为基础的大小排除色谱，我们将分离和表征核糖体组装复合物和snRNP复合物的组成部分，在纯化的细胞核和纯化的核核提取物中，从对照细胞分离，或从不同的NB调节剂处理的细胞，使用基于质谱的蛋白质组学和RNAseq。我们将结合荧光和电子显微镜进行分子分析，分析影响核仁、CBs和斑点之间蛋白质和RNP复合物的运输、方向性和运动速率的机制。

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.