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

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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.