Unravelling the modular architecture of the Ccr4Not-complex

The development of systems biology has revealed that cells use complex functional networks, to control all aspects of life, including responses to the external environment. Perturbation of these networks can leads to too little or too much growth of the cells or even to cell death. Control and fine-tuning of the diverse networks in response to environmental changes is accomplished by adjusting the abundance of the individual proteins according to the actual needs of the cell.

One obvious way to do this is to change the levels of their corresponding mRNAs. The Ccr4Not complex, global player in the regulation of the mRNA levels acts at both ends of the spectrum of mRNA life. On the one hand, it can target the RNA polymerase II to genes to produce new copies of mRNAs. On the other, it can prepare selected mRNAs for degradation. In order to ensure that appropriate adjustments are made, the Ccr4Not-complex senses the state of the cell by communicating with reporter proteins. According to the reports received, the complex switches its functionality and regulates mRNA levels either positively or negatively.

Today, we understand very little about how the Ccr4Not complex accomplishes this fascinating role as master control complex. Some evidence suggests that it has different modules, which it can activate, deactivate, add or delete. Yeast has at least two Ccr4Not complexes: a smaller core complex and a larger complex whose various attachments provide additional functionalities.
I propose to explore where the modules and the attachments are placed and how they switch the functionality of the complex. We will use electron microscopy to visualize the different variants of the complex followed by computational analysis to reconstruct their three-dimensional shapes. The resulting structures will tell us how the different modules are arranged in respect to each other and may provide insight into the mechanisms underlying their regulation of mRNA levels.

The different variants of the Ccr4Not complex can either promote degradation of mRNA or promote generation of mRNA. These different processes occur at different places in the cell. We will use advanced light microscopy to map the distribution of the different variants of the Ccr4Not complex in cells and determine how this changes under different growth conditions.

As a master controller, the Ccr4Not complex is vital for the health of cells and organisms. This is especially evident in stress situations, where cells with impaired Ccr4Not complexes fail to grow properly. At the organismal level, defects in the Ccr4Not complex can lead to heart disease or influence the outcome of certain types of cancer. Indeed the links between function of the Ccr4Not-complex and human health are only now beginning to emerge. Therefore, a mechanistic understanding of structure/function relationships in the Ccr4Not complex will yield important insights, both for a basic understanding of how cells interact with their environment, but also for human health.

The Ccr4Not complex regulates gene expression by mechanisms conserved from yeast to humans. Some of the regulatory functions of the Ccr4Not-complex are at the transcriptional level and probably linked to the core promoter binding whereas others involve the deadenylation of the poly(A)-tail of mRNA, which is a signal for mRNA-degradation. Current evidence suggests that the Ccr4Not-complex has a modular architecture, which enables it to change its function. In yeast the Ccr4Not complex exists in two variants, with approximately 1 MDa and 1.9 MDa, respectively. Despite the important regulatory role of the Ccr4Not complex, structural insights into the organization of this complex regulator are few, and further study is imperative. I hypothesize that changes in the modular organisation of the complex may help cells to adapt to different growth conditions. I therefore propose to use electron microscopy and image processing to study the architecture of the core complex and its larger variant. These investigations will reveal how the two variants are organized, where the subunits and modules in the complexes are located and how the core-complex changes its conformation upon substrate binding. These structural studies will be complemented by biochemical and biophysical characterization of the binary protein-protein and protein-substrate interactions using surface plasmon resonance measurements and state-of-the-art mapping of crosslinks between members of the isolated complexes by mass spectrometry. Characterization of the binary interactions will reveal the network of interactions between subunits in the Ccr4Not complex, their contribution to the stability of the complex and identify subunits that contribute to substrate binding. Finally, live cell imaging will map the distribution of the large and small variants of the Ccr4Not-complex in cells and reveal how it changes during the cell cycle and in response to the nutritional state of the cell.

Who will benefit from the research?
The immediate beneficiaries are in the academic sector, and include students in Biology and Molecular Medicine, researchers in Cell-Biology, RNA-Biology and molecular Electron Microscopy. In longer terms, the research might become important for pharmacologists, who want to exploit the regulatory role of the Ccr4Not-complex in controlling heart disease and certain cancers. Further indirect beneficiaries are members of the School of Biological Sciences at the University of Edinburgh (UoE) and SULSA (Scottish University Alliance for Live Science).

How will they benefit from this research?
The outcome of the research will increase the general understanding on how levels of mRNA are controlled in the cell. This is a fundamental question in Biology, which is vital to health and disease. Therefore, research in this area will update the text book knowledge, which is taught to students in Cell Biology, Molecular Biology and molecular Medicine.
Researchers in Cell Biology and RNA-Biology will benefit from a structural model of the Ccr4Not complex, which will be important for informing experiments to probe the function of the complex in a rational way.
A link between heart disease and the malfunction of a component of the Ccr4Not complex was established in 2010. This demonstrates the global impact that the disturbance of the Ccr4Not complex has on the whole organism. Other studies suggest links between other components of the Ccr4Not-complex and colorectal cancer. Due to this involvement in different types of diseases, it appears likely that the Ccr4Not complex will emerge as an important drug target in future. The exploitation of this drug target would greatly benefit from detailed structural insights into the organization of the complex.
Members at the School of Biological Sciences at the UoE will benefit from the local implementation and improvement of baculovirus expression. The proposed project will increase the local critical mass of expertise and thus will help to start further projects using similar methods quicker and more cost-effective.
Users of the High-Resolution Cryo Electron microscopy Facility at the UoE from across Scotland will gain from the project-based implementation and enhancement of high-resolution data acquisition and single particle image analysis.
The project is divided in several smaller sub-projects, which offer potential for the training of research students at the cutting edge of technology in electron microscopy, complex expression and protein-protein interaction studies. In the long-term, this will produce highly trained individuals, with a good portfolio of experimental methods that will enable them to tackle new projects from different methodological angles.

What will be done to ensure that the beneficiaries benefit from this research?
The scientific results of this research will be communicated to the beneficiaries by publication in peer reviewed journals, by deposition of the data in appropriate data bases and by the presentation of the results in national and international meetings.
Technological advances established in the EM-facility at the UoE will be communicated to other facility-users in local users meetings, Wiki-style protocols and project based experimental advice supported by the SULSA technologist (Chris Kennaway). Chris will also be involved in the project, which will enable an effective transfer of knowledge between different projects in the facility.