Evaluating the mechanisms that drive the displacement of dsRNA-binding proteins from dsRNA

The growth, function and death of cells in multi-cellular organisms need to be tightly controlled. Key to this are the mechanisms for accessing and utilizing the information stored in our genomes. Correct cellular behaviour is dependent on this information being used at the right time and in response to the appropriate stimuli. Malfunctions in regulatory mechanisms can mean that this information is no longer or incorrectly accessed. Such mutations often lead to uncontrolled cell growth and function, which are the hallmarks of cancer. An accurate and precise understanding of the cellular mechanisms that control the use of genomic information is thus a pre-requisite for understanding how diseases like cancer develop and propagate.
One recently identified regulatory system, which controls many different cellular processes, involves the biological polymer RNA, a molecular relative of DNA. A large array of short regulatory RNA molecules has been identified including many that are implicated in cancers. These short RNA molecules have been termed microRNAs (or miRNAs) due to their comparatively small sizes. Cells produce the majority of these miRNA molecules via the same system. This process is performed by a number of molecular machines. The research outlined in this proposal aims to characterize the mechanisms by which cells produce small RNA regulators.
One aspect of this system that is particularly interesting is that some of the protein factors that contribute to the production of regulatory RNAs are also employed in other cellular processes. In this application we plan to determine how these proteins multi-task and how other protein factors facilitate or inhibit the individual tasks. There is a body of research that suggests that some viruses target the protein factors that make microRNAs to alter their ability to multi-task.
One of the cornerstones of molecular biology is the effort to understand cellular processes at the atomic and molecular levels. This presents many technical challenges. The molecules of interest need to be prepared carefully and analyzed with sophisticated equipment. The goal of this proposal is to reveal atomic resolution pictures of the molecular process of miRNA production, to combine this precise and accurate structural data with information about molecular movement, and to relate understanding from the atomic level with that at the cellular level. To achieve this requires a multidisciplinary approach and the use of sophisticated experimental strategies.
Why is this level of understanding important? To have a comprehensive understanding of a biological process, or how a disease affects it, requires knowledge at every biological level, from physiology to biochemistry to molecules and atoms. Important information about the role of molecular structure in the processes described here is currently missing from our understanding. Such information is extremely valuable, both in terms of our fundamental understanding of the process of disease and in our attempt to design and produce molecular therapies. The research outlined here will continue to reveal atomic resolution insights into the factors that define, control and regulate the production of regulatory RNAs in humans. Understanding the common and unique themes could, for example, help scientists design molecules that selectively inhibit the synthesis of RNA regulators that implicated in the development of diseases.

Double-stranded (ds) RNA has a vital role as an intermediate in processes crucial to normal cellular function as well as being an intermediate in the replication of certain viruses. The related dsRNA-binding proteins, TRBP and PACT, bridge two fundamental RNA pathways in humans: the biogenesis of microRNAs and the detection of viral dsRNA. TRBP and PACT bind miRNA precursors as well as interacting with Dicer, a key enzyme in miRNA biogenesis. TRBP and PACT also interact with Retinoic acid-inducible gene I (RIG-I) and dsRNA activated protein kinase R (PKR), two viral dsRNA sensors that are components of the innate immune response to viral infection. In published and preliminary work, we have demonstrated that homodimerisation of TRBP or PACT uses the same interface required for interaction with Dicer; and that TRBP/PACT bind non-specifically to dsRNA, forming higher order complexes that conceal the Dicer binding surface. Dicer must therefore liberate two interfaces: it must dissociate a protein/protein interface and it must displace TRBP/PACT from its target dsRNA. How this happens is unknown.
RIG-I and PKR also interact with both dsRNA and PACT/TRBP. Despite evidence for these interactions in the literature, it is not known whether these associations are mediated by direct protein/protein interactions or bridged by indirect protein/RNA interactions. There is also little molecular-level information about the interfaces involved.
Here we propose to explore how effector proteins like Dicer, RIG-I and PKR facilitate the displacement of TRBP/PACT from target dsRNAs. We will elucidate the 3D structure of the homodimeric dsRBD that Dicer must dissociate and which our preliminary data indicate dimerises by a novel mechanism. We will evaluate the specific protein- and dsRNA-interactions that drive homodimerisation of TRBP and PACT and determine how these interactions are modulated by Dicer, PKR and RIG-I, and factors such as phosphorylation or viral proteins.

This proposal concerns basic mechanistic research into fundamental cellular processes. We envisage that our results will have wide-ranging impact on research in associated areas. We also foresee longer term impact in the following areas:

> Impact on Health

Changes in miRNA function are associated with a broad spectrum of diseases and chronic conditions, including cardiovascular disease, fragile X syndrome and DiGeorge syndrome. The growing catalogue of diseases associated with miRNAs is documented at the Human MiRNA and Disease Database (http://www.cuilab.cn/hmdd). Furthermore, there are myriad examples of miRNAs functioning as oncogenes or tumour suppressors. Mutation to TRBP has been reported in certain cancers and reduced levels of Dicer in cancer cells correlates with poor outcome. Our understanding of the interplay of the various proteins that contribute to pre-miRNA processing by Dicer is poor, particularly the roles of PACT and TRBP. The research outlined here will evaluate how TRBP and PACT interact with pre-miRNAs and engage with Dicer. Our research will allow the functions of to the two accessory proteins to be more precisely defined.
TRBP and PACT are also implicated in the cellular response to viral infection. The data generated by our research will underpin longer-term studies into therapies targeted to controlling viral infection and viral regulation of the innate immune response. In addition, by investigating interactions between TRBP/PACT and Dicer, PKR and RIG-I we can evaluate the interplay between these two pathways, which is currently not well understood.
Mutations in PACT are associated with dystonia through changes in the PACT interaction profile and the activation of effector proteins like PKR. Our research will provide a framework for analyzing the link between mutation of PACT and changes in the interaction profile. Again, these data will drive future studies into this condition as well as possibly identifying new routes to therapy.

>Impact on Education

The project will provide an excellent opportunity for the PDRA to gain first-hand experience in the interdisciplinary process of structural molecular biology research. This project will utilize multiple research techniques that together report on a broad range of biological scales, from atoms to cells. The integration of structural and molecular biology techniques is now critical for projects that aim to address complex cellular and molecular processes. There is strong push for UK research labs to train interdisciplinary scientists. Modern PDRAs need to be familiar with a range of techniques, know how to design integrated research strategies, and know how present the data in a way that is understandable to a broad scientific audience. Participating in this project will therefore allow the PDRA to gain valuable experience in these skills.
The named research technician (MH) will be trained in the production of recombinant proteins using a baculoviral expression system. MH already has strong expertise in protein and RNA production. Participation in this project will further broaden his skill set thereby enhancing his employment possibilities at the end of the project.
A number of undergraduate and research students will contribute to the project, which will provide a fantastic opportunity to perform cutting-edge research into vital and exciting cellular systems. The PDRA will co-supervise undergraduate students, thereby gaining valuable experience in devising and directing research projects.