Programme Leader: Professor Anne Willis
Control of translation is one of the major mechanisms of gene expression regulation. This process can be modulated both globally and for specific regulation of subsets of mRNAs under defined cellular conditions, including toxic injury, and the data suggest that over 90% of all mRNAs have the capacity to be controlled in this way. Many eukaryotic mRNAs are primarily regulated at the level of translation and these include those that have cellular functions that are associated with cell proliferation, cell death and tumourigenesis. This is unsurprising since modifying the rate of synthesis of a protein allows a cell to respond rapidly to changing conditions without the requirement for the production of new mRNA.
The majority of translational regulation occurs at the level of initiation, a highly integrated process that in mammalian cells requires canonical initiation factors (eIFs), sequence elements within the 5’ and 3’ untranslated regions of the mRNA and trans-acting proteins and RNAs. Many mRNAs are translated by a mechanism that has been termed cap-dependent scanning, which requires the binding of the trimeric complex eIF4F to the 7-methyl G cap structure and scanning to the first AUG codon that is in good contex. However, the ability of the ribosome to be recruited to and scan along the RNA is influenced by sequence elements within both the 5' and 3’ untranslated regions of the mRNA. Elements present within the 5’-UTR that regulate translation include internal ribosome entry segments , upstream open reading frames and terminal oligopyrimidine tracts (TOPs; whereas regulatory elements within the 3’-UTR include sequences that control the translation, localisation and stability of an mRNA (e.g. ZIP motif and oligopyrimidine tracts; and micro RNA ((miRNA) target sites Figure 1).
Potential mechanisms for translational regulation: These can involve (i), structured regions or IRESs in the 5’(ii) upstream open reading frames or (iii) or miRNA target sites in the 3’ UTR.
Cytoplasmic post-transcriptional control mediated through these mRNA elements is tightly regulated by defined groups of mRNA binding proteins and the data suggest that specific ‘RNA-operons’, comprised of RNA motifs and their cognate RNA binding proteins, ensure co-regulated synthesis of proteins with common functions, Sawicka et al; 2010 under review). In this regard has been shown that regulation of the levels/phosphorylation status of cytoplasmic RNA-binding proteins directly influences cellular fate under different conditions, again demonstrating the importance of cytoplasmic regulation in the overall control of gene expression. For example, during apoptosis there is an increase in the cytoplasmic levels of PTB that leads to activation of IRESs found in mRNAs that encode proteins that are required for this process to proceed; a reduction in PTB levels alone is sufficient a decrease in the apoptotic rate.
Post-transcriptional profiling of gene expression demonstrates that there is a reprogramming of protein synthesis following toxic cellular insult mediated by a range of different agents (including UV exposure, cisplatin and TNF related apoptosis inducing ligand, TRAIL). This reprogramming event occurs in addition to transcriptional control, although in comparison to transcription control post-transcriptonal regulation has been little studied. From the systems studied thus far the data suggest that following toxic injury subsets of mRNAs are co-ordinately regulated and whilst some of the same mRNAs are regulated under different conditions the total changes in the mRNAs that are selectively translated is specific to the type of toxic injury imposed upon the cell. This reprogramming is an essential part of the cellular response to toxic injury stress. Thus by combining post-transcriptional and transcriptional profiling data it will possible to generate new models to accurately predict the effect of exposure to different classes of toxic agents. This programme of work will allow the establishment of an international centre of expertise that will coordinate transcriptional and post-transcriptional toxicology profiling data and support other researchers in the UK toxicology community by providing access to these technologies.
The specific objectives are:
1. To investigate post-transcriptional regulation of gene expression following exposure to a range of toxic agents including doxorubicin, TNF, 5 fluoro uracil: It is necessary to perform multiple different post-transcriptional screens in one well-defined model system. We will perform a) polysome profiling and b) miRNA profiling. Bioinformatics based approaches will be used to ensure that these data are handled and interpreted in a thorough and systematic fashion.
2. To identify regulatory RNA elements which mediate these responses.
We will identify the regulatory elements that are present in these mRNAs in a) 5’ UTRs, b) 3’UTRs and c) combination of 5’ and 3’ UTRs.
3. To analyse the global role RNA binding proteins using a) ribonomics and b) siRNA polysome profiling. c) Animal models will be used to assess the role of these proteins in vivo.
4. To use NHL as a model system in which to study de-regulation of a cytotoxic response.
5. To use a systems-based approach to study the development of disease following toxic exposure using mesothelioma as a model system.
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