Mechanisms of alternative translation initiation codon selection in the regulation of eukaryotic gene expression

The DNA sequence in every cell of the body, termed the genome, stores the instructions to make all the proteins that are the essential building blocks needed for living. The code for each protein is stored within shorter stretches of DNA called genes. Converting the DNA sequence of a gene (which has only four 'letters') to that of its corresponding protein (which is made up of twenty different kinds of amino acids) requires two major processes, transcription and translation. As their names suggest, transcription is the copying of the sequence information in the DNA into a molecule called messenger RNA (mRNA) whereas translation involves the decoding of the mRNA sequence into the amino acid sequence. The complex molecular machine that translates the mRNA sequence is called the ribosome, and the positioning of the ribosome on the mRNA sequence requires several other proteins, termed translation initiation factors. The ribosome starts making a protein when it finds a particular sequence in the mRNA called a translation initiation codon. Originally it was thought that each gene only made one protein but it is now well established that multiple proteins can be produced from a single gene. This makes a lot of sense when we consider that the genomes of several animal species have now been sequenced and the human genome only contains between 25,000 and 30,000 genes, yet humans are a much more complex animal than a fruitfly (which has 13,600 genes) or a nematode worm (19,000 genes). Different proteins can be made from the same gene if the transcription process begins at different places within the gene or if the mRNA is edited differently in a process called alternative splicing. Another way in which different proteins can be produced from the same gene is by the ribosome beginning to translate the protein at different positions on the mRNA, meaning that longer or shorter versions of the protein are made. I am interested in how and why different translation initiation codons are used and just how widespread this phenomenon is. So far, there are only a few examples where this has been discovered but those that are known are very important, making proteins which can have completely different functions or go to different places within the cell. Importantly, most of the examples of proteins that use alternative initiation codons are implicated in cancers, suggesting that making a certain form of the protein might be better for the cell than when an alternative start site is selected. The work I propose to carry out will determine what factors influence the ribosome to start translating a protein at one position versus another. This could be due to certain sequences being recognised in the mRNA, or it could be due to other translation initiation factors being involved in the selection process. I will be using a few examples of proteins that are known to be made from different initiation codons for this work, but the discoveries I make will be useful in improving our understanding of how ALL proteins can be made, both under normal cellular conditions and also when this process goes wrong. We are going to carry out this work by changing the mRNA sequences surrounding the translation initiation codon to see if we can make their selection better or worse. We will also manipulate the levels of the initiation factors in different cell lines that we grow in the laboratory to see if having more or less of these factors also has an effect on initiation codon choice. Another part of the project will monitor which particular versions of proteins are being made in patient samples, and we may therefore be able to use our knowledge of what role the versions play in the cell to begin to improve the diagnosis of diseases.

The regulation of eukaryotic translation initiation is a key point in gene expression, and can rapidly alter the temporal and spatial expression of either the whole transcriptome or specific mRNAs, without requiring new transcription. Central to translation initiation is the selection of the translation initiation codon (TIC), usually an AUG. However, other AUG and non-AUG codons may be used for translation and I intend to investigate the usage of alternative initiation codons and how these result in the synthesis of N-terminally extended or truncated proteins. My preliminary work has demonstrated that the open reading frame of the multi-domain translation initiation factor eIF4GII can be extended by over 140 amino acids through the use of a non-canonical CUG initiation codon, in addition to the originally assigned AUG. This project will expand this work to determine the mechanisms of alternative TIC selection using the examples of eIF4GII, eIF4GI (translated as 5 isoforms from alternative AUGs), and BAG-1 (with 3 isoforms, the longest from a CUG). Using a simple assay I have developed, I will demonstrate the usage of alternative TICs on these mRNAs and confirm translation occurs from these codons and other non-canonical triplets by mutagenesis. Regions of primary sequence or secondary structure that influence initiation codon choice will also be examined. Quantitative RT-PCR and immunoblotting will relate the different protein species arising from alternative TICs to the endogenous mRNA. Further work will examine the role of trans-acting factors in the selection of initiation codons using a combination of overexpression and siRNA. Signalling events that impinge upon translational machinery will also be examined to determine whether particular kinases control TIC choice. This work will begin to examine the importance of alternative TIC selection in the generation of protein isoform diversity, a previously neglected aspect of gene expression.

This proposal is the beginning of several long term projects I am undertaking to examine the expansion of the proteome by use of alternative translation initiation codons, which will have major implications in the areas of biotechnology (in particular proteomics studies) and human health. This work will make us reconsider mechanisms of gene expression and I believe that alternative initiation codons may represent as important a method for generating diversity in the proteome as alternative splicing. This work will benefit those working in the proteomics community as the expansion of the proteome due to use of alternative translation initiation codons is not often considered in such studies. For example, peptides arising from mass spectrometric analyses of a protein with multiple translation initiation codons (e.g. eIF4GI) may not be assigned correctly as they will be truncated compared to the expected peptide size. Information regarding the use of alternate initiation codons, including estimates of their usage in different tissues (see below) will therefore be useful in performing such analyses, so will be made available in public gene expression databases. I am currently engaged in work to identify further candidates which are likely to be expressed as different isoforms from alternative translation initiation codons. This work will therefore be of interest to workers in many fields, not just gene expression. As this work expands, the physiological roles of such different isoforms will be explored and work by others has recently shown that alternate isoforms of the two pore domain potassium (K2P) channels TREK1 and TREK2 that arise from different translation initiation codons show altered biophysical properties and ion selectivity. A long-term objective of this research is to determine whether the use of alternative initiation codons can be used as a biomarker to determine the status of gene expression in diseased tissues. The groundwork for this approach has already been proven for BAG-1, one of the examples that will be studied during this project. Increased nuclear staining of BAG-1 in breast cancer (correlating with increased usage of the most 5' non-canonical CUG initiation codon) is associated with improved long-term prognosis. Being able to assay a patient sample for different isoforms of a protein that arise from alternative initiation codons where the isoforms have defined properties may enable diagnoses to be made. One simple way in which this could be performed would be a simple ELISA test, if suitable antibodies to different isoforms could be developed. There is already a large and successful infrastructure in place at the University of Southampton to exploit novel biological research in the clinical environment. This, coupled with the extensive networking opportunities afforded to me in the newly built Institute for Life Sciences and as an active member of the Southampton Nucleic Acids Partnership and the South Coast RNA Network as well as the wider Translation Control community will ensure that I am able to disseminate this work to the largest audience possible and create strong collaborations in the future. This work is also amenable to therapeutic interventions and prospective collaborations with industrial partners will be sought at the earliest stages of this project, in order to create the biggest potential impact in improving quality of life and generating economic success.