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Proteomic Analysis of Translation Initiation in Yeast

酵母翻译起始的蛋白质组学分析

基本信息

批准号：
8035791
负责人：
MICHAEL P WEIR
金额：
$ 32.82万
依托单位：
WESLEYAN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-20 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8035791
关键词：
Accounting Affect Algorithms Alternative Splicing Bioinformatics Biological Assay Biological Models Biology Candidate Disease Gene Codon Nucleotides Complementary DNA Complex Data Data Set Disease Epitopes Eukaryota Event Future Gene Expression Profile Gene Expression Regulation Gene Proteins Genes Goals Individual Initiator Codon Lead Mass Spectrum Analysis Messenger RNA Methods Molecular Cloning Molecular Genetics Mutation N-terminal Open Reading Frames Organism Peptides Plasmids Play Prevalence Process Proteins Proteome Proteomics RNA Splicing Research Ribosomes Role Saccharomycetales Site Students Terminator Codon Testing Transcription Initiation Site Transgenes Translation Initiation Translations Variant Yeast Model System Yeasts base computer science disease phenotype experience graduate student improved in vivo research study tandem mass spectrometry yeast protein

项目摘要

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.

描述（由申请人提供）：酵母基因的标准符号表明，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，最近的蛋白质组学规模的核糖体分析数据，以及生物信息学的结果表明，翻译可以启动在目前没有注释为起始位点的mRNA上的网站。这种潜在的翻译起始复杂性增加了令人兴奋的可能性，即在翻译起始水平上的基因调控可能比以前认识到的发挥更大的作用。本项目的主要目标是以芽殖酵母为模型系统，评估由不同翻译起始事件贡献的基因的蛋白质产物库。第一个目的是通过N-末端肽选择方法表征蛋白质N-末端的复杂性。将使用串联质谱法分析源自蛋白质N-末端的胰蛋白酶肽序列。光谱的SEQUEST解释将结合预测的替代翻译起始位点以及预测的翻译后切割事件。该分析将评估在注释的起始密码子的上游和下游的AUG密码子或非规范（nonAUG）密码子处的翻译起始的普遍性。第二个目的是评估选择的注释的ORF使用羧基标记的蛋白质的替代起始位点。将对基因的羧基标记的初级蛋白产物进行部分纯化和分析，以使用质谱法确定其N-末端。将检测具有潜在错误注释的翻译起始位点的单个基因。将使用着丝粒质粒中的表位标记的转基因进行突变分析，以确定注释的或新涉及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中基因的蛋白质产物。随着未来深度测序实验中预期的mRNA序列的大数据集，翻译起始预测的改进将使识别预期影响翻译起始和改变蛋白质产物的序列变体和突变变得更容易，包括改变翻译起始导致疾病表型的情况。) 公共卫生相关性：基于最近的研究，包括转录组规模的核糖体分析，很可能在翻译起始水平的基因表达调控比以前认识到的更重要，我们计划通过质谱和分子遗传学方法相结合，在酵母模型系统中研究这一点。对酵母和高等生物中基因翻译起始事件库的进一步了解将提供更全面的蛋白质组注释，并且将特别有助于对未来mRNA序列的大数据集进行功能解释，特别是在分析与疾病状态相关的mRNA序列变体和突变时。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Assessment of MS/MS search algorithms with parent-protein profiling.

DOI：
10.1021/pr401090d
发表时间：
2014-04-04
期刊：
JOURNAL OF PROTEOME RESEARCH
影响因子：
4.4
作者：
Lin, Miin S.;Cherny, Justin J.;Fournier, Claire T.;Roth, Samuel J.;Krizanc, Danny;Weir, Michael P.
通讯作者：
Weir, Michael P.

Amino termini of many yeast proteins map to downstream start codons.