Analysis of the mechanism for coupled translation in cellular and virus mRNAs

Translation is the final stage of the most fundamental process in biology in which the genetic material of the organism is turned into proteins. During the process of translation; ribosomes (which are the cellular machines that build proteins) make the new protein by following a plan written in a molecule called mRNA. mRNA is a single stranded molecule made up from 4 different components (called nucleotides) which act like letters in our alphabet to make a code. Scientists have been studying the way the ribosome machine works in eukaryotes (higher organisms) for many years and thought that all proteins are made in the same way. Viruses are a group of microscopic organisms which infect and cause diseases in organisms. During the infection process they must use the infected cell's apparatus including ribosomes to make more new viruses. Viruses have been forced to exploit all of the cell systems to survive and multiply faster before the infected cell can destroy it. Importantly, all strategies used by the virus must be compatible with the workings of the cell to function. Interestingly, by studying these new strategies Scientists have seen that not only, do the viruses use them, but the cells also do. We are studying a virus called respiratory syncytial virus (RSV) which will have likely infected most individuals a child and will have a good chance of infected them again as adults. The RSV virus also uses a novel mechanism to express a protein called M2-2. Here instead of just making one protein from a piece of mRNA two proteins are made. We are interested in working out how this is possible. We have shown that in order to make the second protein: M2-2 the first protein M2-1 must be made. This means the ribosomes must go in reverse as the coding sequence for the second protein overlaps with the coding sequence of the first. This was an important finding as ribosomes had not previously been shown to have this ability. Important regions within this mRNA have been discovered that allow the second protein to be made. These regions appear to be present throughout the coding sequence of M2-1. If we change these regions by introducing mutations or make deletions the amounts of the second protein made are reduced, in some cases completely, indicating their importance. These sections contain a lot of secondary structure. Secondary structure occurs where the nucleotides in different parts of the mRNA molecule can interact together, which can be thought of as like fitting two pieces of a jig-saw together. As more pieces are added the structure gets stronger. We are going to use the latest techniques to capture and pinpoint where the ribosome is on the M2-1 RNA. If the ribosome is in one location more than by random chance this will indicate the location where the ribosome has paused. This could 1) allow the ribosome to effectively think and change its mind. So instead of doing what it should and following a signal to stop and move away it re-starts and makes a new protein and or 2) The secondary structures inhibit ribosome movements so that gaps appear on the mRNA molecule so a ribosome stopping can also move back and not be blocked. We are also interested in finding out if other proteins assist in this process. We aim to fish out these proteins using our M2-1 RNA as bait. We have also discovered for the first time that our genome contains examples of mRNA that carry out this process. We have at least 5 examples to investigate further and highlights the universal use of this mechanism.

The eukaryotic translation machinery is able to control gene expression by exploiting a number of alternative processes which determine the frequency with which specific mRNAs are translated. We described the first example of a controlled translation reinitiation event in the M2 mRNA of human RSV in which ribosomes translate the RSV M2 gene second ORF by reinitiating upstream of the ORF-1 termination codon: expression from ORF-2 requires prior termination of ORF-1 translation. A putative regulatory sequence has been identified upstream of the site of the coupling process. We will study the mechanism of coupled translation in the M2 mRNA, by extending preliminary data showing that cellular proteins bind to the regulatory region in a sequence-specific way. Proteins in purified RNA protein complexes will be identified by mass spectrometry. Using a ribosomal profiling technique we will determine the precise location of ribosomes on the M2 mRNA and on mutated mRNAs unable to direct the coupling process to show whether ribosome stalling or pausing is a necessary component for coupling. This will be complemented by an analysis of the structure of the mRNA in the regulatory region to identify residues involved in extensive base-pairing. We have also demonstrated coupled translation in 5 cellular mRNAs. Our data indicates that the mechanism of coupling in these mRNAs may differ in detail from other systems. We will explore this further using mutagenesis of a sequence repeat motif which we have shown to be important for coupling to occur. We will determine if this repeat is inherently essential or if it can be substituted by alternative repeat motifs. To determine if ribosome pausing is a common feature in the coupling process we will conduct ribosome profiling to focus on the five candidate genes we have already identified. We will also use a bioinformatic approach to look for additional mRNAs in the transcriptome that show evidence of ribosomes in the 3' UTR.

The study of the control of gene expression is a fundamental aspect of cell and molecular biology. Gene regulation is central to all of the major biological processes such as cellular development, cell division and metabolism as well as the processes which can lead to abnormalities such as cellular transformation and cancer. In particular, gene regulation at the level of the translation has been demonstrated to be essential for a range of important processes, for example in the early stages of embryonic development and in vascularisation of tumours in whole animals. The work in the project will investigate a novel translational control process which will undoubtedly be involved in a similar array of important cellular processes. The approach to be taken in the project will utilise a genome-wide analysis and is anticipated to identify a large number of new candidate genes which display translational regulation. As a direct consequence the work will also identify previously unknown gene products for further investigation. The project will therefore be of interest not only to the scientific community with a specific interest in translational regulation but also to the wider community with interest in gene regulation in the broadest sense. The wider community also includes scientists in the commercial sector who exploit knowledge of control mechanisms to design drugs that target regulatory processes. The analysis that the project will conduct will explore the detailed mechanism of coupled translation in the M2 gene of human respiratory syncytial virus (RSV). RSV is the major cause of hospitalisation of infants less than one year of age worldwide. RSV also infects adults where the impact is a high level of morbidity which has social and economic consequences. No vaccine or therapeutic is available to prevent or treat RSV infection in humans. The proteins encoded by the M2 gene carry out important regulatory functions in the RSV replication cycle and perturbing their expression is likely to have significant adverse effects for the virus. We anticipate that this study will provide insights which may be of value to researchers in the academic and commercial sectors seeking new avenues to ameliorate the impact of RSV disease in the human population. We are aware that the ribosomal profiling approach will generate a dataset of extreme value to researchers in virology and general cell biology by providing not only the complete translational profile of the virus but also of the infected and uninfected cell and of the impact of infection on translational control of host gene expression. We will ensure that this data is made available to the scientific community in a timely and open way. An important component of the work is the development of new computer programmes to aid genome analysis. In particular, the project will lead to the development of new techniques for data mining and data presentation for the newly described technique of ribosomal profiling. This technique, and therefore the tools developed to maximise its utility, has potential interest across a vast range of scientists. We intend to make the datasets we generate available through publicly accessible web-based forums. The data will be presented in easily accessed formats to encourage exploitation by the greatest number of researchers.