Comprehensive Discovery of Functional Elements of the Drosphila Transcriptome

我们的主要目标是全面鉴定果蝇转录组的功能元件，包括Poly(A)-转录本、所有翻译区域和所有与蛋白质结合的RNA元件。此外，我们还建议阐明调节RNA剪接、翻译和稳定性的RNA序列和结合蛋白。我们计划在整个发育过程中调查具有代表性的时间点，调查各种组织类型和特征良好的细胞系。我们将制作一个单碱基分辨率的Poly(A)转录本目录，并使用RLM-RACE验证转录起点和终点的子集。我们将使用核糖体图谱发现mRNAs和长非编码RNA的新翻译元件，并使用蛋白质表达分析验证子集。我们将使用亲和纯化和高通量测序(RIP-SEQ)来全面鉴定果蝇中由完整的RNA结合蛋白(RBPs)结合的RNA，优先考虑人类同源物。限制性商业惯例识别并结合特定的RNA序列，这些相互作用可以调节mRNAs的稳定性、剪接、核输出、亚细胞定位和可译性。在这些研究的同时，生物信息学分析将识别新的Poly(A)转录本，模拟其二级结构，发现新的mRNAs翻译元件，如uORF和识别长的非编码RNA。对于每个RNA结合蛋白(RBPs)，我们将使用重复性、生物复制数和相对丰富的统计指标来识别结合的转录本。我们还计划确定结合位点基序(PWMS)，并发展RNA-蛋白质相互作用网络。我们将把RNA-蛋白质相互作用图与出版的果蝇蛋白质相互作用图整合在一起。 这些研究的范围是史无前例的，将为任何生物体的转录后基因调控提供最全面的一套实验证据。作为一种公共资源，这些研究是了解正常新陈代谢、生长和分化的先决条件，这将有助于了解其他生物以及人类健康和疾病中的这些过程。

Our primary goal is to comprehensively identify the functional elements of the Drosophila transcriptome, including the poly(A)- transcripts, all translated regions, and all RNA elements bound by proteins. In addition, we propose to elucidate the RNA sequences and binding proteins that regulate splicing, translation and stability of RNA. We plan to survey representative time points throughout development, a wide-variety of tissue types and well-characterized cell lines. We will produce a catalog of poly(A)- transcripts with single-base resolution and validate a subset of transcription start sites and termination sites using RLM-RACE. We will discover novel translated elements of mRNAs and long non-coding RNAs using ribosome profiling and validate a subset using a protein expression assay. We will comprehensively identify the RNAs bound by the complete set of RNA-binding proteins (RBPs) in Drosophila using affinity purification followed by high throughput sequencing (RIP-seq), prioritizing human homologs. RBPs recognize and bind to specific RNA sequences, and these interactions can regulate the stability, splicing, nuclear export, subcellular localizatin, and translatability of mRNAs. Concurrent with these studies, bioinformatic analyses will identify new poly(A)- transcripts, model their secondary structures, discover novel translated elements of mRNAs such as uORFs and identify long non-coding RNAs. For each RNA-binding protein (RBPs), we will identify bound transcripts using statistical measures of reproducibility, biologica replicates and relative enrichment. We also plan to identify binding-site motifs (PWMs) and develop the network of RNA-protein interactions. We will integrate the RNA-protein interaction map with the published Drosophila Protein Interaction Map. The scope of these studies is unprecedented and will provide the most comprehensive set of experimental evidence for post-transcriptional gene regulation in any organism. As a public resource, these studies are a prerequisite for understanding normal metabolism, growth and differentiation that will aid in understanding these processes in other organisms and in human health and disease.