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

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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.