The role of poly ADP ribosylation in mRNA export

In order for the cells of an organism to live they need to synthesise proteins. Within cells a special template called messenger RNA (mRNA) 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 holes in it called nuclear pores. The mRNA is transported from the nucleus, through the nuclear pores to the cytoplasm of a cell which surrounds the nucleus. In the cytoplasm mRNA is decoded by transfer RNA (tRNA) to make proteins and this process is essential for cell survival. A complex of proteins called TREX determines when a mRNA is fully processed and ready to leave the nucleus and then helps transport it to the cytoplasm. We have found that when DNA is damaged, the proteins within TREX are modified in a special way and this appears to enhance the way that TREX can assemble in the cell. We do not yet know why this happens to TREX but we suspect that it may allow the cell to make special proteins required to repair damaged DNA. In this project we will dissect how DNA damage modifies the activity of TREX and how the mRNAs required to survive DNA damage are transported from the nucleus to the cytoplasm in the cell. These studies are important because the DNA in our cells is constantly being damaged and must be repaired if we are going to live a long healthy life. 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, production of biopharmaceuticals and understanding what keeps humans healthy.

PolyADP ribosylation (PARylation) of proteins is a well known response to DNA damage and other cellular stresses such as heatshock. Recent proteome wide studies have identified numerous new targets for PARylation and we noted that numerous mRNA export factors including multiple TREX subunits are targets for PARylation. Strikingly, a key peptide which we previously showed was important for association of key TREX subunits is frequently PARylated and we have shown that this modification stimulates the interaction between key subunits of TREX. We have also found that TREX subunits associate with a protein which is selectively recruited to BRCA1 responsive genes and therefore we hypothesise that through this recruitment, TREX stimulates the export of mRNAs required to recover from DNA damage. PARylation of TREX subunits may help this complex function in a selective way in times of stress. In this proposal we aim to 1) establish genome wide picture of which mRNAs are exported during a DNA damage response 2) determine what effect PARylation has on the activity of TREX and the export receptor heterodimer Nxf1:Nxt1 3) Examine how TREX might be selectively recruited to key mRNAs required to recover from cellular stress such as DNA damage. We will use a range of molecular biology techniques to tackle these questions in this project and address fundamental and important questions about how genes are expressed in eukaryotic organisms.

The work addresses the basic biology of how genes are expressed in human cells, how this is modulated in response to DNA damaging agents and the role that polyADP ribosylation plays in this process. There are three main groups of beneficiary from this work 1) Clinicians and cancer patients. PARP inhibitors are now licensed for clinical use for the treatment of ovarian cancer. Our studies will uncover the role that PARylation plays in regulating mRNA export in normal cells and during DNA damage and how this might help normal cells repair damaged DNA. PARP inhibitor treatments have the potential side effects of triggering secondary cancers. In the long term our studies may help identify suitable biomarker assays which could be used to monitor patients during PARP inhibitor treatments to minimise the possibility of patients developing secondary cancers. 2) Researchers engaged on the project. The project will provide new training opportunities for the people engaged on this project and this in turn will develop their careers and contribute to the generation of a highly skilled workforce which is essential for the continued success of the U.K. economy. 3) The general public/schoolchildren. Media training will be utilised during the project to enhance the ability of the investigator to deliver an accessible educational message to the general public about the various research projects being undertaken in his laboratory and those of colleagues working in related areas in the University. This will help enhance the public understanding of science which is increasingly important, particularly with imminent large scale sequencing of human genomes through the 100,000 genome project. A series of courses will be run for schoolchildren to experience molecular biology techniques and they will be given a specific talk describing the recent research of SW, including work in this project and how this is contributing to our understanding of human cells and how they work. This will be important for inspiring schoolchildren to become the next generation of scientists in the future.