Investigation of the translational regulation of terminal oligo pyrimidine (TOP) containing mRNAs

Our bodies' cells are built from a diverse set of building blocks called proteins. All proteins can be made by translating specific pieces of the DNA genetic code. An intermediate material called mRNA is used between DNA and proteins; it presents the parts of the genetic code for proteins needed at a particular time and place to the machinery that translates it into proteins. The 'molecular factory' that translates mRNA into proteins is called the ribosome, made itself of a mixture of about 80 RNAs and proteins. Without ribosomes, no proteins could ever be made. When our cells grow and divide, the number of each of the component parts of ribosomes also has to double so that enough proteins can continue to be made. Incorrect proportions of component parts of the ribosome can cause cells not to grow enough, or to grow too much, and so imbalances can lead to diseases such as cancer. The many proteins whose job is to work as part the ribosome have a unique 'tag' (called a TOP or terminal oligopyrimidine tract) in the mRNA that encodes them. All mRNAs with this tag have their translation closely co-ordinated by existing ribosomes so that their proteins are made at the same time and rate. While we know about the existence of the tag, we do not fully understand how it is recognized and used to control new ribosome manufacture. Our lab has done initial experiments which reveal a unique suite of proteins that bind to the tag. In this application we propose to study exactly how these proteins interact with the tag, and with each other, to co-ordinate ribosome manufacture so accurately. Our results could help us to understand how cancers develop when this goes wrong, and, in the longer term, could provide new ways to tackle this disease.

The coordinated synthesis of ribosomal proteins and parts of the translational apparatus is absolutely essential to cell growth and division and even small perturbations in the relative levels of one of these proteins can result in cell cycle block, apoptosis or if unchecked can lead to inappropriate cell division and tumorigenesis. These proteins are all encoded by mRNAs which contain an unusual feature in the 5' untranslated region, a terminal oligopyrimidine tract (TOP) of 7-14 nucleotides that immediately follows the 7-methyl G cap at the 5' end of the mRNA. It is clear that the presence of this sequence element permits a unique method of translation of this subset of mRNAs and they display a binary 'all or none' association with polysomes that is dependent on the cell state. Thus in the presence of serum they are associated with polysomes whilst following serum starvation these mRNAs rapidly dissociated and instead are found present in subpolysomal particles. This is significantly different from non -TOP-containing mRNAs whose distribution between polysomes and subpolysomal particles in much less well defined. In terms of abundance TOP-containing mRNAs comprise 30% of the total cellular mRNAs in a growing cell, however little is known about how they are co-ordinately regulated or how these sequences associate with the 40S ribosomal subunit. Our preliminary data suggest that TOP-containing mRNAs have a lower requirement for canonical initiation factors than other mRNAs and that they interact with a unique complex of proteins. We propose to extend our initial work to identify the full complement of proteins which interact with TOP-containing mRNAs, to investigate how these regulate TOP-mRNA translation and to determine how these sequence elements act to recruit the ribosome.

Who will benefit? The results of the proposed research will have important implications for understanding how a subset of mRNAs is co-regulated and for ribosome biogenesis. This project will also be important for the understanding of gene regulation by control of translation, which has a broad impact on cellular growth, development and cancer. The impact of the results of this research will extend beyond the scientific community, with potential for commercialisation leading to economic benefits. In the longer term, benefits to general public health and are likely to result. The research has the potential to engage the interest of the general public and avenues for engagement with the public, the media and policy makers will be pursued. How will they benefit? The proposed research will result in an increased understanding of the mechanisms by which TOP-elements can operate in the regulation of gene expression, and the factors that govern these mechanisms. TOP-containing mRNAs have a central role to play in cell growth, and changes in TOP-regulation are associated with cancer. An understanding of the factors that regulate TOP-mRNA translation will present new avenues for modulation of these important regulators, and potentially for therapeutics. The postdoctoral scientist and research assistant employed on the project will develop vital skills in written and oral communication to a range of audiences, project and time management, and critical and creative thinking. They will be expected to engage both with the broader scientific community and the general public, and to undertake formal training where relevant. Plans for engagement An important aspect of any research is education of the public. In this respect an important aspect is to engage the general public about the science behind our work. The applicant has a good track records in disseminating science and are regularly involved in Cambridge Science week. Kathryn Lilley regularly takes visiting students from elsewhere in the UK and the EU into her lab for periods of time to teach them technologies in place in her lab. She will continue to do this and hence the methodologies developed and applied here will be disseminated to many other research labs in Europe. She also teaches on several external courses including the Masters' programmes at the Nottingham Trent University, Cranfield University, Leicester University and York University. Technologies developed here will also be relayed to students during these lectures.