Unr - master regulator of mRNA translation

All cells in our body need to be able to turn genes on and off at the right times; it is important to know how this is controlled, in order to understand processes such as development and aging. Many genes function together in the same processes within a cell, so need to be turned on, or expressed, together. To achieve this, there are some proteins that control many genes at once - master regulators. One protein that we believe acts in this way is called Unr. Mice that do not have Unr are not viable - the embryos die during development - which shows that this protein is crucial for the functioning of a healthy animal. Our preliminary studies have shown that Unr can stimulate the expression of many, but not all, genes. Although we know that Unr is very important, and we know that it has a role in gene expression, we do not know: 1) which genes Unr turns on, or 2) how it does this. The objectives of this project are to find answers to these two questions. Firstly, we will use an antibody that recognises Unr to isolate the protein from human cells, along with all the different genes that it is interacting with. We will then be able to identify those genes, and the specific parts of those genes that mark them out to be turned on by Unr. We will also compare healthy cells with cells that do not have Unr, and look to see which genes are no longer expressed in the absence of Unr. Secondly, we will carry out experiments to test which other proteins Unr interacts with in order to stimulate expression of a gene. We already know some of the proteins that Unr interacts with, but not whether these interactions are important for its function in gene expression, or how these interactions enable Unr to turn a gene on. In unravelling the function of the master regulator Unr, this project will increase knowledge of the complicated regulation of gene expression in human cells, and help to shed light on how the human body develops and ages.

Gene expression is controlled post-transcriptionally through editing, splicing, nuclear export, localisation, translation and degradation of mRNAs. Groups of mRNAs whose protein products are involved in the same cellular processes are co-ordinately regulated through the binding of specific RNA-binding proteins. These 'RNA operons' are themselves likely to be co-ordinately controlled by 'master regulators' - RNA-binding proteins that bind to and stimulate or inhibit the expression of a number of RNA operons. We and others have shown that the RNA-binding protein Unr regulates the expression of many genes at the post-transcriptional level, and we propose that Unr is a master regulator of translation. Genetic experiments in mice have shown that Unr is crucial for development and correct cellular regulation. The first objective of this project is to identify the RNA operons whose expression is regulated by Unr, using the methods of ribonucleoprotein-immunoprecipitation followed by microarray analysis (RIP-chip), and translational profiling; and to determine the sequence, location and multiplicity of RNA motifs recognised by Unr in the mRNAs that it binds, using the recently described photoactivatable-ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP). The second objective of this project is to investigate the mechanism by which Unr stimulates the translation of the mRNAs to which it binds. Initiation of translation depends on the interactions between the 5' and 3' ends of an mRNA, including those between the cap-binding protein eIF4E, eIF4G, and the poly(A) tail binding protein PABP1. We will characterise the interactions between Unr, PABP1 and eIF4G, and also between Unr, the eIF4E binding protein 4E-T, and eIF4E to determine how Unr regulates the translation of the mRNAs to which it binds. The two complementary strands of this project will make a significant contribution to our knowledge of post-transcriptional control of gene expression.

All cell and molecular biology is underpinned by studies on gene expression. The importance of controlling translation is being increasingly recognised; translational control has been shown to play a key role in processes such as development, metabolism, cell signalling and nerve transmission. A number of RNA-binding proteins have been identified that control expression of certain genes involved in these processes. We have identified an RNA-binding protein that controls translation of a large subset of genes, and in this project we propose to identify the genes that it controls and how it does so. We also believe that genes are regulated in a combinatorial manner, and that the combination of proteins that bind to an mRNA controls its expression. This project therefore seeks to make a major contribution to the understanding of coordinated gene expression. As such, the results will be of interest to a great number of scientists who study the regulation of gene expression, both in academia and industry. Knowledge of how genes are regulated is crucial to our basic understanding of the function of the human body, and has the potential in the future to contribute to the design of novel gene-based therapies. It is known that knocking out the protein we are studying in mice leads to lethality during embryogenesis, and that embryonic stem cells from the mice have defects in the apoptotic response to DNA damage. Therefore, it is likely that the results of our research will impact on the fields of development and aging. These are areas of intense investigation by both the public sector and pharmaceutical companies, and we anticipate that our results will be of great interest to both. In the long term, the results of this project may be useful for maintaining the health of both the developing foetus and the aging body, and so have benefits for health and welfare in the UK and internationally. A further benefit of this project is provided by the bioinformatics software tools that will be developed to analyse the genes that are identified as being regulated in our experiments. These will be of interest to researchers worldwide studying the regulatory codes of many other organisms, e.g. plants and fungi, thus contributing to research in areas as diverse as food security and bioterrorism.