Dynamics of the nucleolar transcriptional machinery

• Applicant: Professorin Dr. Ingrid Grummt
• Subject Area: Biochemistry
• Term: from 2002 to 2007
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5367014

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.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1050: Funktionelle Architektur des Zellkerns