Dynamics of the nucleolar transcriptional machinery

核仁是细胞核区隔化和基因表达空间组织的范例。在这个细胞器中，核糖体RNA基因被转录，前RNA被加工，前核糖体颗粒被组装。尽管对核糖体合成的各个步骤进行了广泛的分析，但对活细胞中这些过程的空间和时间动力学知之甚少。本项目主要研究核仁转录机制的时空分布，以及调控转录和核仁结构的调节蛋白复合物。我们将解决以下问题：Pol I转录机制如何对核（ol）ar结构做出贡献？在细胞分裂和生长因子信号传导过程中，核仁蛋白复合物是否易位到其他细胞室？核仁转录机制是如何在有丝分裂结束时精确定位的？为了回答这些问题，我们将建立表达多种gfp标记核蛋白的细胞系，并将使用FRAP/FLIP方法来研究细胞周期不同阶段和不同生长条件下活细胞内蛋白质的定位、相互作用和迁移。荧光相关光谱（FCS）将测量大分子相互作用的动力学参数。最后，我们将在哺乳动物细胞中采用RNA干扰（RNAi）敲除基因的方法，研究个体蛋白在体内对核仁结构和功能的作用。我们期望这些快速和非侵入性的技术将揭示活细胞中核仁蛋白复合物的动力学和生化特性，并有助于揭示核仁结构和功能的相互依存关系。

The nucleolus is a paradigm for nuclear compartmentalization and spatial organization of gene expression. In this organelle ribosomal RNA genes are transcribed, pre-rRNA is processed and preribosomal particle are assembled. In spite of extensive analyses of individual steps of ribosome synthesis, little is known as to the spatial and temporal dynamics of these processes in living cells. This research project focuses on the temporal and spatial arrangement of the nucleolar transcription machinery as well as on regulatory protein complexes controlling transcription and nucleolar structure. We will address the following questions: How does the Pol I transcription machinery contribute to nucle(ol)ar architecture? Are nucleolar protein complexes translocated into other cellular compartments during cell division and growth factor signaling? How is the nucleolar transcription machinery targeted to accurate locations at the exit of mitosis? To answer these questions, we will establish cell lines that express diverse GFP-tagged nucleolar proteins and will use the FRAP/FLIP approach to study protein localization, interactions and mobility within a living cell both in distinct phases of the cell cycle and under different growth conditions. Kinetic parameters of macromolecular interactions will be measured by fluorescence correlation spectroscopy (FCS). Finally, we will use the method of gene knock-out by RNA interference (RNAi) in mammalian cells to study the role of individual proteins on nucleolar structure and function in vivo. We expect that these rapid and non-invasive techniques will reveal the dynamics and biochemical properties of nucleolar protein complexes in the living cell and will help to uncover the interdependence of nucleolar structure and function.