Defining the Xenopus translatome

非洲爪蟾翻译组的定义

• 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
• 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

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）： 摘要本申请描述了发育生物学家Michael Sheets博士和生物统计学和生物信息学专家Colin Dewey博士之间的多学科合作，Michael Sheets博士专注于早期非洲爪蟾胚胎发生的转录后调控，Colin Dewey博士专注于使用深度测序方法分析基因表达。虽然从转录的角度理解非洲爪蟾的发育已经取得了进展，但对mRNA转录物翻译成蛋白质的了解却少得多。我们建议使用核糖体分析全球范围内测量非洲爪蟾mRNA的翻译活性，从卵子发生到原肠胚形成的发展窗口。从拟议的实验结果将提供一个前所未有的动态翻译景观，在非洲爪蟾的发展过程中存在的，这个数据有许多应用。首先，我们的实验将确定生物学相关的爪蟾mRNA。转录组分析只能识别细胞中存在的mRNA，但不能确定哪些mRNA被翻译成蛋白质，因此具有生物学相关性。我们将使用核糖体分析，以确定积极翻译和生物学相关的非洲爪蟾mRNA。mRNA群体的这个子集，特别是那些表现出调节的mRNA群体，代表了功能丧失研究的理想候选者。其次，我们的研究结果将确定组的mRNA表现出类似的模式的翻译调控。这些共同调节的mRNA将为分子研究提供重要的起点，这些分子研究试图鉴定介导调节的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下的核糖体分析将使我们能够鉴定我们样品中每个Xenpus mRNA的翻译起始位点。起始位置定义了由mRNA编码的蛋白质产物的氨基末端，因此全局鉴定所有mRNA的起始位点定义了蛋白质组的N-末端。此外，识别特定mRNA的起始位点， 可以揭示5' RNA序列的存在，称为uORF，其通常用于调节翻译起始。从拟议的实验结果将提供一个前所未有的基因组规模的非洲爪蟾的mRNA翻译分析，以及如何翻译的每个mRNA的变化在发展过程中。我们的研究结果和相关的方法将是有用的爪蟾研究人员和研究人员解决同样的问题，在其他脊椎动物胚胎。