Genome-wide analysis of polyadenylation and translation in Xenopus

描述（申请人提供）：非洲爪蟾（Xenopus laevis）和热带爪蟾（Xenopus tropicalis）是研究脊椎动物发育、生殖细胞生物学和细胞周期控制各方面的主要模式生物。从历史上看，由于易于培养，体积大，以及生化纯化材料的可用性，X. laevis一直是首选的模型蛙。然而，部分由于X. laevis的异源四倍体性质，直到最近它还缺乏一个测序和注释的基因组。最近发布的非洲爪蟾基因组序列草案促进了全基因组方法在这一重要模式生物中基因表达研究的应用。通过测量特定时间点的转录本水平来测定基因表达的全基因组方法在识别对许多过程重要的五个因素方面非常成功。然而，对特定转录物稳态水平的了解并不等同于对该转录物中蛋白质表达时间的精确了解。在像非洲爪蟾这样的生物中，早期发育是通过母体mrna的翻译来编程的，在没有转录的情况下，将RNA丰度的测量与翻译输出的测量结合起来尤为重要。在这个提议中，我们描述了一种新的mrna测序策略的使用，该策略可以识别在卵子发育晚期和早期发育时转录沉默时显示调控表达模式的mrna。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。本研究将利用新颖的方法和非洲爪蟾最新的基因组序列来深入了解基因表达的转录后调控，并利用这些信息来确定关键发育时间点的重要因素。最后，该提案将为整个爪蟾群落产生许多资源，并确保爪蟾在未来成为关键的模式生物。

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.