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

Lead Research Organisation: University of York
Department Name: Biology


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.

Technical Summary

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.

Planned Impact

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.
Description We have determined that some of the key protein molecules involved in the biosynthesis of small regulatory RNA molecules in humans have an usual way of interacting with each other. Most molecules self-associate in a symmetrical way; that is a simple rotation can map one molecule onto another in a multimeric assembly. Our data shows that two proteins important in the production of human regulatory RNAs self-associate in an asymmetric manner. We have extensive data to support this finding, which was published in Nucleic Acids Research in 2017. In 2017/2018 we have elucidated the 3D structures of the homodimerisation domains of TRBP and PACT using X-ray crystallography. This work will submitted for publication in 2018. Knowledge of the molecular processes that underpin how these protein molecules associate together and with each other is vital for understanding how the cell regulates the production of regulatory RNA molecules and for the development of molecular tools and therapeutics that can target the process.
Exploitation Route Understanding how protein molecules that participate in the biosynthesis of regulatory RNAs interact is vitally important to a more general understanding of this pathway, its function in the cell, and how this function can be manipulated, both by natural biological processes and by human intervention.
Sectors Pharmaceuticals and Medical Biotechnology

Description DL 
Organisation Hull York Medical School
Country United Kingdom 
Sector Academic/University 
PI Contribution a
Collaborator Contribution a
Impact 1. Conserved asymmetry underpins homodimerization of Dicer-associated double-stranded RNA-binding proteins. Heyam A, Coupland CE, Dégut C, Haley RA, Baxter NJ, Jakob L, Aguiar PM, Meister G, Williamson MP, Lagos D, Plevin MJ. Nucleic Acids Res. 2017 Dec 1;45(21):12577-12584. PMID: 29045748 2. Argonaute Utilization for miRNA Silencing Is Determined by Phosphorylation-Dependent Recruitment of LIM-Domain-Containing Proteins. Bridge KS, Shah KM, Li Y, Foxler DE, Wong SCK, Miller DC, Davidson KM, Foster JG, Rose R, Hodgkinson MR, Ribeiro PS, Aboobaker AA, Yashiro K, Wang X, Graves PR, Plevin MJ, Lagos D, Sharp TV. Cell Rep. 2017 Jul 5;20(1):173-187. doi: 10.1016/j.celrep.2017.06.027. PMID: 28683311 3. S6K2-mediated regulation of TRBP as a determinant of miRNA expression in human primary lymphatic endothelial cells. Warner MJ, Bridge KS, Hewitson JP, Hodgkinson MR, Heyam A, Massa BC, Haslam JC, Chatzifrangkeskou M, Evans GJ, Plevin MJ, Sharp TV, Lagos D. Nucleic Acids Res. 2016 Nov 16;44(20):9942-9955. Epub 2016 Jul 12. PMID: 27407113 4. Dissecting the roles of TRBP and PACT in double-stranded RNA recognition and processing of noncoding RNAs. Heyam A, Lagos D, Plevin M. Wiley Interdiscip Rev RNA. 2015 May-Jun;6(3):271-89. PMID: 25630541
Start Year 2012
Description Regensberg 
Organisation University of Regensburg
Country Germany 
Sector Academic/University 
PI Contribution Communicated data and experimental results
Collaborator Contribution Communicated data and experimental results. Sent constructs to York for experiments
Impact Conserved asymmetry underpins homodimerization of Dicer-associated double-stranded RNA-binding proteins. Heyam A, Coupland CE, Dégut C, Haley RA, Baxter NJ, Jakob L, Aguiar PM, Meister G, Williamson MP, Lagos D, Plevin MJ. Nucleic Acids Res. 2017 Dec 1;45(21):12577-12584. PMID: 29045748
Start Year 2017
Description Sheffield 
Organisation University of Sheffield
Department Department of Molecular Biology and Biotechnology
Country United Kingdom 
Sector Academic/University 
PI Contribution Prepared samples for analysis in Sheffield
Collaborator Contribution Access to NMR equipment and expertise
Impact Conserved asymmetry underpins homodimerization of Dicer-associated double-stranded RNA-binding proteins. Heyam A, Coupland CE, Dégut C, Haley RA, Baxter NJ, Jakob L, Aguiar PM, Meister G, Williamson MP, Lagos D, Plevin MJ. Nucleic Acids Res. 2017 Dec 1;45(21):12577-12584. PMID: 29045748
Start Year 2014
Description Ducker 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Participated in discussions with poet Christy Ducker and film maker Kate Sweeney who were creating a body of work around the concept of "messengers" which culminated in a book and poetry reading and film presentation in a gallery in York.
Year(s) Of Engagement Activity 2017
URL https://www.york.ac.uk/c2d2/projects/alive/#tab-2
Description Pint of Science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact PDRA on BBSRC award BB/N018818/1 (Dr Clement Degut) presented a public lecture on protein structural biology using X-ray crystallography as part of the Pint of Science festival.
Year(s) Of Engagement Activity 2018
URL https://pintofscience.co.uk/event/revolutionary-science