Mechanistic determination of how microRNAs control gene-expression

Recently, a completely new way of controlling gene expression has been discovered. This has come to light after the identification of a new class of genes, which unlike most genes do not produce proteins, but instead are processed into short RNA molecules called microRNAs. There are around 1000 different microRNAs within the human genome, all of which have different effects. They work by binding to the messenger RNA of protein encoding genes and inhibiting production of the protein encoded within the messenger RNA. Each of these 1000 small RNA molecules is believed to interact with and regulate around 200 protein encoding genes, thus adding to the complexity of the regulation of the human genome. Already it has become clear that malfunction of miRNA regulation is associated with virtually all human disease, including: cancer, diabetes, and viral infections. In 2002 Science magazine called miRNA the breakthrough of the year, and these small RNA molecules have been termed the "Dark Matter of the cell". MicroRNAs were only discovered in 2001 and, amazingly, already within this short period, microRNA-based drugs are in clinical trials for a number of human diseases demonstrating the usefulness of the research within this field.
However, despite the rapid advances within this field, how these small RNA molecules exert their effects on protein production is currently unclear and controversial. As manipulation of these small RNA molecules is a realistic approach for the treatment of a number of human diseases, understanding how these therapeutic agents work will be critical for their development and safe use.
This proposal aims to determine the mechanism by which these small RNA molecules control the production of proteins within the human body and to resolve the controversy within the field by supplying testable models which can be probed by many laboratories around the world.

The discovery of microRNAs (miRNAs) has revolutionised the way we view the control of gene expression. MiRNAs are small RNA molecules that bind to complementary sites within the 3`UTR of target mRNAs and negatively regulate gene expression. It is clear that miRNAs induce both translational repression and accelerate mRNA decay; however, the contribution of these two mechanisms has been greatly debated, as has the mechanism of translational repression. Recently a number of groups, including ours, have shown that translational repression occurs first and involves inhibition the eIF4F translation initiation complex by evicting the DEAD-box RNA helicase eIF4A. In parallel to these observations, we and others have shown that the Ccr4-Not complex via its central component cNOT1 interacts with two different DEAD-box RNA helicases, namely DDX6 and eIF4A2 and this leads to translational repression.
This application outlines a series of complementary experimental approaches that will combine to comprehensively address how miRNAs control gene expression. It will determine the role of the DEAD-box RNA helicases within the repression pathway and identify whether they act in parallel or at different stages of repression. It will examine whether the DEAD-box RNA helicases are associated with miRNA-targeted mRNAs at different stages of the repression pathway. It will also identify the differential protein composition of the repression complexes containing different DEAD-box RNA helicases. Currently it is unclear how the association of DDX6 and eIF4A2 with cNOT1 alters their activity and leads to the eviction of the helicase from eIF4F. Experiments outlined within this proposal will identify how these complexes interact and lead to the inhibition of protein synthesis. Finally we will determine the atomic structures of these repression complexes and use this information to examine the repression mechanism.

Who will benefit from this research?
This project focuses on the basic science underpinning gene expression and its control in multicellular eukaryotes and will directly impact human health and disease. In the short term, the main beneficiaries of this research are academic within fields of gene regulation, functional genomics, systems biology and RNA biology, as well as more distantly related fields that focus on various physiological processes important for health and disease. Since miRNAs have been shown to be disregulated in a host of human diseases, the pharmaceutical-industry in the UK and worldwide has invested heavily in developing miRNA-based therapeutics. Some of these compounds are now in clinical trials and early results are very optimistic. However, the full impact of these drugs cannot be assessed until we understand the mechanisms by which they function. This application is aimed at determining this fundamental knowledge, which will have a direct impact on these therapeutic compounds. Other beneficiaries include the biotechnology and pharmaceutical sectors, businesses and institutions in other sectors where highly skilled people are required.

How might they benefit from this research?
This project focuses on determining the basic mechanism by which miRNAs operate and will give great insight into how the major deandenylation complex within eukaryotes organisms functions to control gene expression. A detailed mechanistic understanding will provide a framework for researchers studying the role of gene regulation in relation to miRNAs and mRNA turnover. For example, identification of sequence motifs within mRNAs that allow them to be regulated by miRNAs and the Ccr4-Not complex will be instrumental in unlocking the full scope of these regulatory mechanisms. This has the potential to impact all fields of research studying multicellular eukaryotes. For example, miRNAs in plants have also been shown to operate via translational repression in a similar manner. Defects within the miRNA biogenesis pathway have been strongly linked to a number of human disease states, particularly cancer, but an incomplete understanding of how miRNAs function prevents us from fully exploring their impact. In summary, the outcomes of the proposed work will have a host of implications both in relation to plants, animals and humans, particularly in connection with human health and disease. Currently, together with MRCT, we are developing specific inhibitors of the translation initiation factors eIF4A, and the knowledge and structural information gained from the work proposed in this application may be of great use for the development of these compounds. Thus, this application has the potential to impact on drug discovery and research programmes in academic and industrial settings and the quality of life and wellbeing in the longer term, with benefits to the wider public.

To ensure that the impact of the work will reach researchers in closely and more distantly related fields, we will ensure that we will widely disseminate our research findings via national and international conferences, symposia and high quality peer-reviewed scientific journals using open-access options. High-throughput datasets will be made available via freely accessible repositories. Materials and reagents will be made available as widely as possible. In addition, we will produce
review article(s) to maximise the impact of this project and to reach researchers in more distantly related fields.
The project will result in the training of highly-skilled researchers, who are essential for research and development activities in the pharmaceutical and biotechnology sector. Transferable skills will include large-scale data handling which can be useful in many industries. The biotechnology sector may also benefit from the research via, for example, the licensing of reagents (e.g. antibodies). Novel insights may also benefit other sectors such as publishing and teaching.