Proteomic Analysis of Translation Initiation in Yeast

描述(由申请人提供)：酵母基因的标准符号表明，大多数mRNAs产生由简单的长开放阅读框架(ORF)定义的单一蛋白质产物。然而，最近蛋白质组规模的核糖体图谱数据以及生物信息学结果表明，翻译可以在目前未被注释为起始位点的mRNAs上的位点开始。翻译启动的这种潜在的复杂性增加了一种令人兴奋的可能性，即在翻译启动水平上的基因调控可能发挥比之前认识到的更大的作用。这个项目的主要目标是以发芽酵母为模型系统，评估由不同翻译起始事件贡献的基因蛋白质产物的库。第一个目的是通过N-末端多肽选择方法来表征蛋白质N-末端的复杂性。来自蛋白质N-末端的胰蛋白酶多肽序列将使用串联质谱仪进行分析。对光谱的SEQUEST解释将包括预测的替代翻译起始位置以及预测的翻译后切割事件。这项分析将评估在带注释的起始密码子的上游和下游的AUG密码子或非规范(非AUG)密码子的翻译起始的普遍性。第二个目的是使用羧基标记的蛋白质来评估选定的注释开放阅读框架的替代起始位点。基因的羧基标记的初级蛋白质产物将被部分纯化并分析，以利用质谱仪确定其N-末端。可能有错误注释的翻译起始点的个别基因将被测试。将使用着丝粒质粒中表位标记的转基因进行突变分析，以确定注释或新涉及的翻译起始点的突变是否干扰标记的初级ORF产物的翻译。提高我们对mRNAs翻译起始事件库的理解将导致更准确的算法来预测和注释酵母和多细胞生物体中基因的蛋白质产物。随着未来的深度测序实验有望获得大量的mRNA序列数据集，对翻译起始的改进预测将使识别预期影响翻译起始和改变蛋白质产品的序列变体和突变变得更容易，包括翻译起始改变导致疾病表型的情况。) 与公共健康相关：基于最近的研究，包括转录组规模的核糖体图谱，在翻译启动水平上对基因表达的调控可能比之前认识到的更重要，我们计划通过结合质谱学和分子遗传学方法在酵母模型系统中进行研究。更好地理解酵母和高等生物中基因的翻译起始事件的谱系将提供更全面的蛋白质组注释，并将特别有助于对未来大规模的mRNA序列数据集的功能解释，特别是在分析与疾病状态相关的mRNA序列变异和突变时。

DESCRIPTION (provided by applicant): The standard notations of yeast genes suggest that most mRNAs give rise to a single protein product defined by a simple long open reading frame (ORF). However, recent proteomic-scale ribosome profiling data, as well as bioinformatics results, suggest that translation can initiate at sites on mRNAs not currently annotated as start sites. This potential increased complexity of translation initiation raises the exciting possibility that gene regulation at the level of translation initiation may play a greater role than previously realized. The primary goal in this project is to assess the repertoire of protein products of genes contributed by different translation initiation events, using budding yeast as a model system. The first aim is to characterize the complexity of protein N-termini through an N-terminus peptide selection method. The sequences of tryptic peptides originating from the N-termini of proteins will be analyzed using tandem mass spectrometry. SEQUEST interpretation of spectra will incorporate predicted alternative translation initiation sites as well as predicted post-translational cleavage events. This analysis will assess the prevalence of translation initiation at AUG codons, or non-canonical (nonAUG) codons, upstream and downstream of the annotated start codon. The second aim is to assess alternative initiation sites of selected annotated ORFs using carboxy-tagged proteins. Carboxy-tagged primary protein products of genes will be partially purified and analyzed to determine their N-termini using mass spectrometry. Individual genes with potentially misannotated translation initiation sites will be tested. Mutational analysis using epitope-tagged transgenes in centromeric plasmids, will be performed to determine whether mutation of annotated or newly-implicated translation initiation sites interferes with translation of the tagged primary ORF product. Improving our understanding of the repertoire of translation initiation events on mRNAs will lead to more accurate algorithms for predicting and annotating the protein products of genes in yeast and multicellular organisms. With large datasets of mRNA sequences expected from future deep sequencing experiments, improved prediction of translation initiation will make it easier to identify sequence variants and mutations expected to affect translation initiation and change protein products, including cases where altered translation initiation gives rise to disease phenotypes. ) PUBLIC HEALTH RELEVANCE: Based on recent studies, including transcriptome-scale ribosome profiling, it is likely that the regulation of gene expression at the level of translation initiation is more important than previously realized, and we plan to investigate this in the yeast model system through a combination of mass spectrometry and molecular genetic approaches. Improved understanding of the repertoire of translation initiation events of genes in yeast and higher organisms will provide fuller annotations of proteomes, and will be particularly useful for functional interpretation of future large datasets of mRNA sequences, especially in analyses of mRNA sequence variants and mutations associated with disease states.