Proteomic Analysis of Translation Initiation in Yeast

描述（由申请人提供）：酵母基因的标准标记表明，大多数mrna产生由简单的长开放阅读框（ORF）定义的单一蛋白质产物。然而，最近的蛋白质组级核糖体分析数据以及生物信息学结果表明，翻译可以在目前未标记为起始位点的mrna上启动。这种潜在的翻译起始复杂性的增加提出了令人兴奋的可能性，在翻译起始水平的基因调控可能发挥比以前认识到的更大的作用。本项目的主要目标是评估由不同翻译起始事件贡献的基因的蛋白质产物库，以出芽酵母为模型系统。第一个目的是通过n端肽选择方法来表征蛋白质n端的复杂性。源自蛋白质n端的色氨酸序列将使用串联质谱法进行分析。光谱的SEQUEST解释将包括预测的替代翻译起始位点以及预测的翻译后裂解事件。该分析将评估AUG密码子或非规范（nonAUG）密码子、带注释起始密码子的上游和下游翻译起始的流行程度。第二个目的是使用羧基标记蛋白评估选定的注释orf的替代起始位点。羧基标记的基因初级蛋白产物将被部分纯化和分析，并使用质谱法确定其n端。翻译起始位点可能有错误注释的个体基因将被测试。在着丝质粒中使用表位标记的转基因进行突变分析，以确定标记的或新牵连的翻译起始位点的突变是否干扰标记的初级ORF产物的翻译。提高我们对mrna翻译起始事件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中基因的蛋白质产物。随着未来深度测序实验中mRNA序列的大量数据集，对翻译起始的改进预测将更容易识别影响翻译起始和改变蛋白质产物的序列变异和突变，包括翻译起始改变导致疾病表型的情况。)

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.