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

身体每个细胞中的DNA序列，称为基因组，储存着制造所有蛋白质的指令，这些蛋白质是生活所需的基本组成部分。每种蛋白质的编码都存储在称为基因的较短DNA片段中。将一个基因的DNA序列（只有四个“字母”）转换成相应的蛋白质（由20种不同的氨基酸组成）需要两个主要过程，转录和翻译。正如它们的名字所暗示的，转录是将DNA中的序列信息复制到称为信使RNA（mRNA）的分子中，而翻译涉及将mRNA序列解码成氨基酸序列。翻译mRNA序列的复杂分子机器称为核糖体，核糖体在mRNA序列上的定位需要几种其他蛋白质，称为翻译起始因子。当核糖体在mRNA中找到一个称为翻译起始密码子的特定序列时，它就开始合成蛋白质。最初，人们认为每个基因只能产生一种蛋白质，但现在已经确定，单个基因可以产生多种蛋白质。当我们考虑到几种动物物种的基因组现已被测序，而人类基因组仅包含25，000至30，000个基因时，这很有意义，但人类是比果蝇（具有13，600个基因）复杂得多的动物。或线虫（19，000个基因）。如果转录过程在基因内的不同位置开始，或者如果mRNA在称为选择性剪接的过程中被不同地编辑，则可以从同一基因产生不同的蛋白质。从同一基因产生不同蛋白质的另一种方式是核糖体开始在mRNA上的不同位置翻译蛋白质，这意味着蛋白质的长度或长度会有所不同。我感兴趣的是如何以及为什么使用不同的翻译起始密码子，以及这种现象有多普遍。到目前为止，只有少数几个例子已经被发现，但那些已知的是非常重要的，使蛋白质可以有完全不同的功能或在细胞内的不同地方。重要的是，大多数使用替代起始密码子的蛋白质的例子都与癌症有关，这表明制造某种形式的蛋白质可能比选择替代起始位点时对细胞更好。我打算进行的工作将确定是什么因素影响核糖体开始翻译蛋白质的一个位置而不是另一个位置。这可能是由于mRNA中的某些序列被识别，也可能是由于选择过程中涉及的其他翻译起始因子。我将使用一些已知由不同起始密码子产生的蛋白质的例子来进行这项工作，但我的发现将有助于提高我们对ALL蛋白质如何产生的理解，无论是在正常细胞条件下还是当这个过程出错时。我们将通过改变翻译起始密码子周围的mRNA序列来进行这项工作，看看我们是否可以使它们的选择更好或更糟。我们还将操纵我们在实验室中生长的不同细胞系中的起始因子的水平，以观察这些因子中的多或少是否也对起始密码子的选择产生影响。该项目的另一部分将监测患者样本中产生的蛋白质的特定版本，因此，我们可能能够利用我们对这些版本在细胞中发挥的作用的知识来开始改善疾病的诊断。

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.