Defining the Xenopus translatome

描述(由申请人提供)： 摘要本申请描述了发展生物学家Michael Sheets博士和Colin Dewey博士之间的多学科合作。Michael Sheets博士专注于非洲爪哇早期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。虽然从转录的角度理解非洲爪哇的发育已经取得了进展，但关于mRNA转录本到蛋白质的翻译却知之甚少。我们建议使用核糖体图谱来全局测量非洲爪哇mRNAs从卵子发生到原肠形成的整个发育窗口的翻译活性。拟议中的实验结果将提供一个前所未有的视角，了解非洲爪哇开发过程中存在的动态翻译景观，这些数据有很多应用。首先，我们的实验将识别与生物相关的非洲爪哇mRNAs。转录组分析只能识别细胞中存在的mRNAs，但它不能定义哪些mRNAs被翻译成蛋白质，因此具有生物学意义。我们将使用核糖体图谱来鉴定主动翻译的和生物相关的非洲爪哇mRNAs。这部分信使核糖核酸群体，特别是那些表现出调节作用的亚群，是功能丧失研究的理想候选者。其次，我们的结果将识别出表现出相似的翻译调控模式的mRNA组。这些共同调节的mRNAs将为分子研究提供重要的起点，这些研究试图确定介导调节的RNA结合蛋白或miRNAs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图谱将使我们能够确定我们样本中每个Xenpus mRNA的翻译起始点(S)。起始位置定义了由mRNA编码的蛋白质产物的氨基末端，因此全局识别所有mRNAs的起始位置定义了蛋白质组的N-末端。此外，确定特定mRNAs的起始位置 可以揭示5‘RNA序列的存在，称为uORF，通常起到调节翻译起始的作用。拟议的实验结果将提供一个前所未有的基因组规模的分析，在非洲爪哇的信使核糖核酸翻译，以及如何在发育过程中每个信使核糖核酸的翻译变化。我们的结果和相关方法将有助于非洲爪哇研究人员和研究人员在其他脊椎动物胚胎中解决同样的问题。

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.