Genome-wide analysis of polyadenylation and translation in Xenopus

描述（由申请人提供）：非洲爪蟾和热带爪蟾是用于研究脊椎动物发育、生殖细胞生物学和细胞周期控制各个方面的主要模式生物。历史上，X。由于易于培养、体积大和生物化学纯化材料的可获得性，所以laevis是优选的模型蛙。然而，部分原因是由于异源四倍体X。laevis直到最近还缺乏测序和注释的基因组。最近公布的非洲爪蟾基因组序列的草案，促进应用全基因组的方法来研究在这个重要的模式生物的基因表达。全基因组方法通过测量特定时间点的转录水平来测定基因表达，在鉴定对许多过程重要的新因子方面非常成功。然而，对特定转录物的稳态水平的了解并不等同于对来自该转录物的蛋白质表达时间的精确了解。在诸如非洲爪蟾的生物体中，早期发育是通过在不存在转录的情况下翻译母体mRNA来编程的，将RNA丰度的测量与翻译输出的测量结合起来是特别重要的。在这个建议中，我们描述了一种新的mRNA测序策略，确定mRNA的表达模式，在卵子发生后期和早期发展时，转录是沉默的。此外，我们将使用最近开发的方法来测量RNA与核糖体的关联，以测量蛋白质表达并提供非编码转录本的列表。最后，我们将利用所获得的信息来鉴定对完成减数分裂很重要的新蛋白质和RNA。该提案将使用新的方法和非洲爪蟾最近的基因组序列，以获得更深入的了解基因表达的转录后调控，并利用这些信息来确定重要的因素的一个关键的发展时间点。最后，这个建议将为整个非洲爪蟾社区产生许多资源，并确保非洲爪蟾是未来的关键模式生物。

DESCRIPTION (provided by applicant): Xenopus laevis and Xenopus tropicalis are major model organisms useful for the study of vertebrate development, germ cell biology, and various aspects of cell cycle control. Historically, X. laevis has been the preferred model frog due to ease of culture, large size, and availability of material for biochemical purifications. However, partly due to the allotetraploid nature of X. laevis it has lacked a sequenced and annotated genome until recently. The recent release of a draft of the Xenopus laevis genome sequence facilitates application of genome-wide approaches to study gene expression in this important model organism. Genome-wide approaches that assay gene expression by measuring the levels of transcripts at particular time points have been very successful at identification of nove factors important for many processes. However, knowledge of the steady state level of a particular transcript does not equate to precise knowledge of the timing of protein expression from that transcript. In organisms such as Xenopus where early development is programmed by translation of maternal mRNAs in the absence of transcription it is especially important to couple measurement of RNA abundance to measurement of translational output. In this proposal we describe the use of a novel mRNA-sequencing strategy that identifies mRNAs that show patterns of regulated expression during late oogenesis and early development when transcription is silent. In addition, we will use a recently developed method to measure RNA association with ribosomes to measure protein expression and provide a list of noncoding transcripts. Finally, we will use the information gained to identify novel proteins and RNAs that are important for the completion of meiosis. This proposal will use novel methods and the recent genome sequence of Xenopus laeivs to gain a deeper understanding of posttranscriptional regulation of gene expression and use this information to identify factors important for a key developmental time point. Finally, this proposal will generate many resources for the entire Xenopus community and ensure that Xenopus is a key model organism in the future.