The assembly and function of the TREX complex

In order for the cells of an organism to live they need to synthesise proteins. Within cells a special template called messenger RNA acts as a blueprint for making proteins. mRNA is produced in a special structure inside the cell called the nucleus, which is encapsulated by a membrane with special holes in it called nuclear pores. The messenger RNA is transported from the nucleus, through the nuclear pores to the cytoplasm of a cell which surrounds the nucleus. In the cytoplasm the messenger RNA is decoded to make proteins and this process is essential for cell survival. A special complex of proteins called TREX determines when a messenger RNA is fully processed and ready to leave the nucleus and then helps transport it to the cytoplasm. The TREX complex has many subunits, some of which we have only just discovered. We do not understand what all the subunits of TREX do nor how they all come together to make the complex and transport the messenger RNA. In this proposal we aim to establish how the TREX complex assembles and examine what each subunit within the complex does. Recent work from other scientists has shown that the TREX complex may play other important functions in the cell such as producing special small RNA molecules called short interfering RNAs which interfere with the synthesis of proteins. Therefore our studies on how the TREX complex assembles and works is likely to help us considerably advance our understanding of how proteins are synthesised in cells. Since the TREX complex is conserved from yeast through to man, our studies are likely to have a broad impact in many areas which affect society including food production and understanding what keeps humans healthy.

The TREX complex plays a central role in eukaryotic gene expression, enabling the export of mRNA from the nucleus to the cytoplasm. Recent work has also identified additional roles for the TREX complex in siRNA production in plants and suppression of antisense RNA production in S.pombe. We have recently shown that one important function of the TREX complex is to open up the TAP mRNA export factor and allow it to bind RNA and this process is likely to be coordinated with RNA processing events. In effect, TREX acts as the molecular timer telling the cell when mRNA is ready to be exported. We have discovered multiple new TREX core subunits in recent work and also shown that the UAP56 RNA helicase must bind ATP to assemble TREX. We have also found that UAP56 may go through multiple rounds of ATP hydrolysis to drive assembly of TREX on mRNA. These new discoveries indicate that the assembly of the TREX complex, which is critical for several facets of eukaryotic gene expresison is more complicated than first imagined. In this proposal we aim to examine the functions of the newly identified TREX components, both in terms of their importance for mRNA export but also their role in TREX assembly. We will also establish the protein:protein and protein:RNA interaction network in TREX and examine in detail what role UAP56 plays in the assembly of existing and new TREX components. In preliminary experiments we have confirmed that two subunits of TREX are sumoylated and we aim to investigate the role of this post-translation modification on TREX assembly and function. Finally we aim to examine where TREX assembly occurs in vivo and how individual TREX components govern the localisation of TREX assembly in vivo.

The range of potential beneficiaries for this project is large. The project will expand our knowledge about TREX which plays a role in gene expression by regulating mRNA export and is involved in the production of siRNAs which trigger RNA interference in plants and animals. The conservation of the complex from yeast to man means our studies in the human system will be applicable across a wide range of other organisms of strategic and economic importance to the BBSRC and U.K.. Improved understanding of the process of RNAi/gene expression should lead to the development of crops more resistant to pests and diseases by generating transgenic plants harboring RNAi expression cassettes active against mRNAs from pathogens. Such plants will be of considerable interest and benefit to the U.K. farming industry and should help in our goal to maintain food security in the future. Similarly the development of transgenic animals resistant to pests and diseases should contribute to food security and will be of considerable interest to the livestock industry, providing the U.K with a significant competitive edge in food production. Our research has already directly had such an impact in that promoters we cloned were used to generate the first transgenic chicken resistant to avian flu recently (published in Science, 2011). Such animals are likely to be a major help in combating pandemic influenza in the future, which will have an impact on the majority of the U.K population. Our continue studies on gene expression will be of benefit to the pharmaceutical industry. RNAi is likely to be a major therapeutic tool in the fight against human diseases and natural processes such as ageing in the future . In order to utilise these tools and manipulate endogeneous RNAi we need to understand the basic mechanisms involved in siRNA production and how genes are normally expressed in humans, which involves mRNA export. Understanding how TREX assembles and functions directly addresses these areas. This research will also impact on the wider public through outreach activities with the public and local schools. for example one of my BBSRC funded PhD students set up an organisation called Science Brainwaves (http://sciencebrainwaves.com/), a charity based in Sheffield with the aim of bringing science, medicine, engineering, mathematics and the social sciences to the masses in fun and interesting ways. It hosts various events from lectures and workshops to hands on science experiments at festivals. I together with the technician + post-doc employed on this grant will be involved with Science brainwaves activities during this project. Through my activities as a course deliverer at the Science Learning Centre (Yorkshire and Humberside) I present the latest findings in my research and those in my field to Science school teachers with the express aim of inspiring their teaching to school children. This activity is likely to have a broad impact on many school children indirectly by bringing their teachers up to date with the latest developments in the life sciences. In particular I focus my courses on the area of gene expression and how it can be manipulated. These activities should indirectly inspire the next generation of scientists which will be vital to the U.K. economic success in the future. Dr Viphakone will be encouraged to participate in the Sheffield research Leader's programme which provides a coherent framework of skills training required to be a research leader. He will also develop his molecular biology skills through training in surface plasmon resonance and cutting edge microscopy techniques. Dr Viphakone and Vicky Porteous will develop communication skills with the public through participation in "Science Brainwaves" activities in scschools and directly with the public. Communication and writing skills will also be developed by Dr viphakone through the preparation of manuscripts and posters for publication and through presentation at conferences.