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、所有翻译区和所有与蛋白质结合的RNA元件。此外，我们建议阐明调控RNA剪接、翻译和稳定性的RNA序列和结合蛋白。我们计划调查整个发育过程中具有代表性的时间点，各种各样的组织类型和特征良好的细胞系。我们将使用单碱基分辨率生成poly(a)-转录本目录，并使用RLM-RACE验证转录起始位点和终止位点的子集。我们将使用核糖体分析发现mrna和长链非编码rna的新翻译元件，并使用蛋白质表达试验验证一个子集。我们将利用亲和纯化和高通量测序（RIP-seq）技术，综合鉴定果蝇全套rna结合蛋白（rbp）结合的rna，优先考虑人类同源物。rbp识别并结合特定的RNA序列，这些相互作用可以调节mrna的稳定性、剪接、核输出、亚细胞定位和可翻译性。在这些研究的同时，生物信息学分析将鉴定新的poly(A)-转录本，建立它们的二级结构模型，发现新的mrna翻译元件，如uorf，并鉴定长链非编码rna。对于每个rna结合蛋白（rbp），我们将使用可重复性、生物重复和相对富集的统计方法来鉴定结合转录物。我们还计划鉴定结合位点基序（PWMs）并开发rna -蛋白质相互作用的网络。我们将把rna -蛋白相互作用图谱与已发表的果蝇蛋白相互作用图谱进行整合。

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.