Genome-wide analysis of polyadenylation and translation in Xenopus

描述(申请人提供)：非洲爪哇和热带非洲爪哇是主要的模式生物，可用于脊椎动物发育、生殖细胞生物学和细胞周期控制的各个方面的研究。从历史上看，莱维氏X.laevis一直是首选的青蛙模型，因为它易于养殖，体型大，而且可以用于生化净化。然而，部分由于莱氏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。最近非洲爪哇基因组序列草案的发布促进了全基因组方法的应用，以研究这种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本水平来分析基因表达，在识别对许多过程重要的新因素方面已经非常成功。然而，了解特定转录本的稳定水平并不等同于准确了解该转录本中蛋白质表达的时间。在非洲爪哇等生物体中，在没有转录的情况下，通过翻译母体mRNAs来规划早期发育，将RNA丰度的测量与翻译输出的测量结合起来尤其重要。在这个建议中，我们描述了一种新的mRNA测序策略的使用，该策略识别在卵子发生后期和转录沉默的早期发育期间显示调控表达模式的mRNAs。此外，我们将使用最近开发的一种方法来测量RNA与核糖体的关联，以测量蛋白质的表达并提供非编码转录本的列表。最后，我们将使用所获得的信息来识别对完成减数分裂至关重要的新蛋白质和RNA。这项建议将使用新的方法和非洲爪哇最新的基因组序列来更深入地了解基因表达的转录后调控，并利用这些信息来确定对关键发育时间点重要的因素。最后，这项提议将为整个非洲爪哇群落产生许多资源，并确保非洲爪哇成为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.