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，我们还打算阐明RNA序列和调节RNA剪接、翻译和稳定性的结合蛋白。我们计划调查整个开发过程中的代表性时间点，各种组织类型和良好表征的细胞系。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验证转录起始位点和终止位点的子集。我们将发现新的翻译元件的mRNA和长的非编码RNA使用核糖体分析和验证一个子集使用蛋白质表达测定。我们将使用亲和纯化，然后进行高通量测序（RIP-seq），优先考虑人类同源物，全面鉴定果蝇中全套RNA结合蛋白（RBP）结合的RNA。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。与这些研究同时，生物信息学分析将鉴定新的poly（A）-转录物，模拟其二级结构，发现新的mRNA翻译元件，如uORF，并鉴定长的非编码RNA。对于每种RNA结合蛋白（RBP），我们将使用重现性、生物学重复和相对富集的统计测量来鉴定结合的转录本。我们还计划确定结合位点基序（PWMs），并开发RNA-蛋白质相互作用的网络。我们将整合RNA-蛋白质相互作用图谱与已发表的果蝇蛋白质相互作用图谱。 这些研究的范围是前所未有的，将提供最全面的实验证据，在任何生物体的转录后基因调控。作为一种公共资源，这些研究是了解正常代谢、生长和分化的先决条件，有助于了解其他生物体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.