The role of nonsense mediated decay in antigenic HLA peptide generation

背景：无意义介导的衰变(NMD)通路最近被认为是癌症免疫治疗的新靶点[1，2，3]，然而NMD与突变表位的HLA呈递之间的基本生物学联系尚不清楚。已经提出了两个相互竞争的假说，第一个假说是必须绕过NMD，这会导致突变蛋白丰度增加，并通过规范的mRNA翻译呈现给人类白细胞抗原。第二个模型提出，在核出口与NMD协同作用后直接发生的非规范“先锋轮翻译”产生抗原肽的主要来源[4，5]。解决这一根本问题具有紧迫的生物学/治疗意义。此外，NMD效率的外显子连接复合体模型对哪些变异体将和不会触发降解提供了一个很差的预测性分类，这给解释单个突变的种系致病和免疫原性潜力带来了挑战。目标：1.建立移码突变蛋白是否产生HLA呈现的新表位Vacaconical mRNA翻译，或“先锋翻译”。利奇菲尔德博士的实验室将利用基因组学、转录学和免疫表位学数据来识别一组已知的框架转移突变蛋白，这些突变蛋白可以触发经过验证的人类白细胞抗原多肽。Carlton博士的实验室将使用活细胞成像技术在体内跟踪这些突变的蛋白质，以建立翻译和人类白细胞抗原呈递的路线，并使用经过调整的特技分析(PMID：25792328)。开发一种改进的预测模型，通过使用饱和基因组编辑方法来分类过早终止密码子突变是否会引发NMD。利用下面的旋转项目产生的设计，将使用CRISPR-CAS9编辑在选定的一组参考基因的长度上的每个氨基酸位置引入提前终止密码子突变。然后将使用荧光报告程序对细胞进行筛选，以确定NMD是否在每个突变位置被触发。然后，利奇菲尔德博士的实验室将开发一种新的机器学习分类器算法，以判断哪些突变会引发NMD，哪些不会引发NMD。

Background: The nonsense mediated decay (NMD) pathway has recently been implicated asa novel target for cancer immunotherapy [1,2,3], however the fundamental biological linkbetween NMD and HLA presentation of mutated epitopes is unclear. Two competinghypotheses have been proposed, the first states that NMD must be bypassed, which henceleads to increased mutated protein abundance/HLA presentation via canonical mRNAtranslation. The second model proposes that the non-canonical "pioneer round oftranslation", which occurs directly after nuclear export in coordination with NMD, generatesthe major source of antigenic peptides [4,5]. Addressing this fundamental question is ofurgent biological/therapeutic relevance. In addition, the exon junction complex model of NMDefficiency provides a poor predictive classification of which variants will and won't be triggerdegradation, creating a challenge in interpreting the germline pathogenic and immunogenicpotential of individual mutations.Goals:1. To establish whether frameshift mutated proteins generate HLA presented neoepitopes viacanonical mRNA translation, or the "pioneer round of translation". Dr Litchfield's laboratorywill utilise genomics, transcriptomics and immunopeptidomics data to identify a set of knownframeshift mutated proteins which trigger validated HLA peptides. Dr Carlton's laboratory willuse live cellular imaging to track these mutated proteins in vivo, to establish the route oftranslation and HLA presentation, using an adapted version of the TRICK assay (PMID:25792328).2. To develop an improved predictive model to classify whether premature termination codonmutations will or won't trigger NMD, by using a saturation genome editing approach. Utilisingthe design generated from the rotation project below, premature termination codonmutations will be introduced at every amino acid position across the length of a selected setof reference genes using CRISPR-Cas9 editing. Cells will then be screened using afluorescence reporter to establish whether NMD was triggered at each mutated position. DrLitchfield's laboratory will then develop a novel machine learning classifier algorithm topredict which mutations do and don't trigger NMD.