PCV - Programme interdiciplinaire en physique et chimie du vivant

Integrated structural biology of rDNA transcription – NUCLEOPOL

Submission summary

Synthesis of the large precursor of the ribosomal RNA is performed by the multimeric RNA polymerase I (RNA Pol I) and represents about 65 % of the transcriptional activity in exponentially growing eukaryotic cells. This essential cellular process is tightly regulated during cell cycle and through the activation of several signalling pathways. Ribosomal RNA synthesis occurs in a defined nuclear sub domain, the nucleolus, and is coupled to RNA maturation processes and sequential pre-ribosome assembly. Transcriptional regulation is associated with a complete nucleolar reorganisation which undergoes major modifications during cell cycle and even disappears during mitosis of metazoan and plant cells. A key player in the spatio-temporal variation of nucleolar structure and organisation is the RNA Pol I itself and its organisation into actively transcribed transcription units. Our aim is to better understand the role of RNA Pol I transcription, rRNA maturation and pre-ribosomal assembly in the structural organisation of the nucleolus. We plan to use a multi resolution approach to describe the structure and the dynamics of the enzyme in various cellular and extra-cellular contexts. In a first step we aim at a molecular description of the yeast Saccharomyces cerevisiae RNA pol I transcription initiation complex. We will use a combination of molecular biology, yeast genetics and biophysical approaches to reconstitute in vitro a functional complex from purified components and to visualize these complexes by cryo electron microscopy (Cryo EM) in order to reconstruct a three-dimensional (3-D) model of this complex in a hydrated state. We will take advantage of a yeast strain that stabilizes an interaction between RNA Pol I and a transcription factor Rrn3 which is critical for initiation complex formation. We previously determined a 3-D model of RNA Pol I at 18 Ǻ resolution by Cryo EM that will be used as reference to probe the structural reorganisations of the enzyme upon initiation and the interaction interfaces with transcription factors and DNA. This information will be correlated with the atomic structure of RNA Pol II to identify the structural domains involved. Functional RNA Pol I initiation factors are currently produced in insect cells and purified to homogeneity. Secondly, we plan to study the 3-D structure of partially purified rDNA transcription units by cryo electron tomography. For this we plan to adapt the 'Miller-spread' method used to visualize transcription units and to use dedicated image analysis approaches to gain insight into the molecular organisation of the transcribing RNA Pol I and its associated ribonucleoproteic particle. Since the position of the transcribing RNA Pol I on the gene measures its temporal state, it will be possible to extract time-resolved information on the coupling between transcription, rRNA maturation and pre-ribosomal assembly. We will take advantage of yeast mutant strains and perform immuno-labeling experiments to further explore the rRNA maturation process. Thirdly we plan to locate these transcription units within the cell to analyze their compaction and their position with respect to the documented ultrastructural sub-domains of the nucleolus. A major effort will be produced to locate RNA Pol I molecules within the nucleolus and we will use advanced methods (high pressure freezing, cryo substitution, cryo sections of frozen hydrated cells) to obtain the best possible structural preservation of the cells. Cryo electron tomomography methods will be employed to reconstruct a 3-D model of the cell section containing the nucleolus. Three independent methods will be used to reveal the RNA Pol I molecules: immunolabeling, yeast mutants with modified RNA Pol I occupancy of the rDNA genes and pattern recognition in cryo tomograms. The distribution of RNA Pol I molecules will reveal the 3-D organisation of rDNA transcription units in their cellular context. Finally the dynamic variations of RNA pol I transcribed loci will be monitored by time lapse optical microscopy in living cells. We will take advantage of the yeast system to design strains in which transcribed genes will be fluorescently tagged in order to monitor the dynamics of chromatin associated with rDNA transcription in vivo and thus to correlate optical microscopy observations with high resolution electron microscopy snapshots of defined functional states. The quantitative analysis of the gene movements will help us to identify the parameters that describe gene diffusion in the nucleolar volume. This system will be used to explore the effect of yeast mutant strains that carry defects in the RNA Pol I transcription apparatus and in rapamycine-induced stress conditions. Altogether the planned experiments will provide an integrated view of the spatial and temporal variations of RNA Pol I transcription units within the nucleolus.

Project coordination

Patrick SCHULTZ (CENTRE EUROPEEN DE RECHERCHE EN BIOLOGIE ET EN MEDECINE - CERBM)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

COMMISSARIAT A L'ENERGIE ATOMIQUE - CENTRE D'ETUDES NUCLEAIRES SACLAY
CENTRE EUROPEEN DE RECHERCHE EN BIOLOGIE ET EN MEDECINE - CERBM

Help of the ANR 500,000 euros
Beginning and duration of the scientific project: - 36 Months

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