Defining the Xenopus translatome

非洲爪蟾翻译组的定义

基本信息

批准号：
8554716
负责人：
Michael D Sheets
金额：
$ 21.59万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-05 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8554716
关键词：
Address Animals Bioinformatics Biometry Cell Cycle Cell Differentiation process Cells Complex Data Development Embryo Embryonic Development Event Exhibits Fertilization Freezing Gene Expression Gene Expression Profile Genetic Transcription Genetic Translation Genomics Harringtonine Individual Initiator Codon Interdisciplinary Study Life Maps Maternal Messenger RNA Measures Mediating Meiosis Messenger RNA Methodology Methods Molecular Molecular Machines Oogenesis Open Reading Frames Pattern Polyribosomes Population Positioning Attribute Post-Transcriptional Regulation Proteins Proteome RNA RNA Sequences RNA-Binding Proteins Rana Regulation Regulatory Element Research Research Personnel Ribosomes Sampling Site Staging Transcript Translating Translation Initiation Translational Regulation Translations Untranslated Regions Xenopus Xenopus laevis Xenopus oocyte deep sequencing egg gastrulation inhibitor/antagonist loss of function nuclease oocyte maturation prospective public health relevance research study xenopus development

项目摘要

DESCRIPTION (provided by applicant): ABSTRACT This application describes a multidisciplinary collaboration between Dr. Michael Sheets, a developmental biologist who focuses on post-transcriptional regulation of early Xenopus embryogenesis and Dr. Colin Dewey, an expert in biostatistics and bioinformatics who focuses on analyzing gene expression using deep sequencing methodologies. While progress has been achieved in understanding Xenopus laevis development from a transcriptional point of view, significantly less is known about the translation of mRNA transcripts into proteins. We propose to use ribosome profiling to globally measure the translational activity of Xenopus mRNAs across the window of development that extends from oogenesis to gastrulation. Results from the proposed experiments will provide an unprecedented view of the dynamic translational landscape that exists during Xenopus development and this data has numerous applications. First, our experiments will identify biologically relevant Xenopus mRNAs. Transcriptome analysis only identifies the mRNAs present in cells, but it cannot define which mRNAs are translated into protein and therefore biologically relevant. We will use ribosome profiling to identify the actively translated and biologically relevant Xenopus mRNAs. This subset of the mRNA population and especially those that exhibit regulation represent ideal candidates for loss of function studies. Second, our results will identify groups of mRNAs that exhibit similar patterns of translational regulation. These co-regulated mRNAs will provide important starting points for molecular studies that seek to identify common sequence motifs for RNA binding proteins or miRNAs that mediate regulation. Third, ribosome profiling in the presence of the inhibitor harringtonine will allow us to identify the translational initiation site(s) for each Xenpus mRNA in our samples. The position of initiation defines the amino termini of the protein product encoded by an mRNA and therefore globally identifying the initiation sites of all mRNAs defines the N-terminus of the proteome. In addition, identifying the sites of initiation for specific mRNAs can reveal the presence of 5' RNA sequences, called uORFs that often function to regulate translational initiation. Results from the proposed experiments will provide an unprecedented genomic scale analysis of mRNA translation in Xenopus and how the translation of each mRNA changes during development. Our results and the associated methods will be useful for Xenopus researchers and researchers addressing the same questions in other vertebrate embryos.

描述（由申请人提供）：摘要该申请描述了 Michael Sheets 博士和 Colin Dewey 博士之间的多学科合作。Michael Sheets 博士是一位发育生物学家，专注于早期非洲爪蟾胚胎发生的转录后调控；Colin Dewey 博士是生物统计学和生物信息学专家，专注于使用深度测序方法分析基因表达。虽然从转录角度理解非洲爪蟾发育已取得进展，但对 mRNA 转录物翻译成蛋白质的了解却少之又少。我们建议使用核糖体分析来全面测量爪蟾 mRNA 在从卵子发生到原肠胚形成的整个发育窗口中的翻译活性。所提出的实验结果将为非洲爪蟾发育过程中存在的动态翻译景观提供前所未有的视角，并且该数据具有许多应用。首先，我们的实验将鉴定生物学相关的非洲爪蟾 mRNA。转录组分析只能识别细胞中存在的 mRNA，但无法确定哪些 mRNA 被翻译成蛋白质，因此无法确定哪些 mRNA 具有生物学相关性。我们将使用核糖体分析来识别活跃翻译且具有生物学相关性的非洲爪蟾 mRNA。这个 mRNA 群体的子集，尤其是那些表现出调节作用的子集，代表了功能丧失研究的理想候选者。其次，我们的结果将识别表现出相似翻译调控模式的 mRNA 组。这些共同调节的 mRNA 将为分子研究提供重要的起点，旨在识别介导调节的 RNA 结合蛋白或 miRNA 的共同序列基序。第三，在抑制剂三尖杉酯碱存在的情况下进行核糖体分析将使我们能够识别样品中每个 Xenpus mRNA 的翻译起始位点。起始位置定义了 mRNA 编码的蛋白质产物的氨基末端，因此全局识别所有 mRNA 的起始位点定义了蛋白质组的 N 末端。此外，确定特定 mRNA 的起始位点可以揭示 5'RNA 序列（称为 uORF）的存在，其通常起到调节翻译起始的作用。拟议实验的结果将为非洲爪蟾的 mRNA 翻译以及每个 mRNA 的翻译在发育过程中如何变化提供前所未有的基因组规模分析。我们的结果和相关方法将有助于爪蟾研究人员和研究人员解决其他脊椎动物胚胎中的相同问题。