Comprehensive Discovery of Functional Elements of the Drosphila Transcriptome

果蝇转录组功能元件的全面发现

• 批准号: 8402420
• 负责人: SUSAN E CELNIKER
• 金额: $ 100万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-21 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8402420
• 关键词: Affinity Chromatography; Animal Model; Binding; Binding Proteins; Binding Sites; Bioinformatics; Biological; Biological Assay; Cataloging; Catalogs; Cell Culture Techniques; Cell Line; Classification; Code; Complex; DNA; Data; Data Analyses; Data Element; Degenerative Disorder; Development; Differentiation and Growth; Disease; Drosophila Proteins; Drosophila genus; Elements; Embryo; Functional RNA; Gene Expression Profile; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Transcription; Genome; Goals; Health; Histocompatibility Testing; Homologous Gene; Human; Indium; Malignant Neoplasms; Maps; Measures; Messenger RNA; Metabolism; Modeling; National Human Genome Research Institute; Nuclear Export; Organism; Poly A; Poly(A)+ RNA; Principal Investigator; Process; Protein-Protein Interaction Map; Proteins; Publishing; RNA; RNA Binding; RNA Sequences; RNA Splicing; RNA Stability; RNA analysis; RNA-Binding Proteins; RNA-Protein Interaction; Regulation; Relative (related person); Reproducibility; Research Infrastructure; Resolution; Resources; Ribosomes; Role; Sampling; Site; Societies; Staging; Statistical Data Interpretation; Structure; Surveys; System; Technology; Time; Tissues; Transcript; Transcription Initiation Site; Transgenic Animals; Transgenic Organisms; Translating; Translations; Validation; base; cost; crosslink; human disease; insight; novel; protein complex; protein expression; research study; tool

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.

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)- 转录物、所有翻译区域以及与蛋白质结合的所有 RNA 元件。此外，我们建议阐明调节RNA剪接、翻译和稳定性的RNA序列和结合蛋白。我们计划调查整个发育过程中的代表性时间点、各种组织类型和特征良好的细胞系。我们将生成具有单碱基分辨率的 Poly(A)- 转录物目录，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 和长非编码 RNA 的新翻译元件，并使用蛋白质表达测定验证一个子集。我们将通过亲和纯化和高通量测序 (RIP-seq) 来全面鉴定果蝇中与整套 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)-转录物，模拟其二级结构，发现新的mRNA翻译元件（例如uORF）并鉴定长非编码RNA。对于每种 RNA 结合蛋白 (RBP)，我们将使用再现性、生物复制和相对富集的统计测量来识别结合的转录本。我们还计划识别结合位点基序 (PWM) 并开发 RNA-蛋白质相互作用网络。我们将把 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整合起来。 这些研究的范围是前所未有的，将为任何生物体的转录后基因调控提供最全面的实验证据。作为公共资源，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体以及人类健康和疾病的这些过程。

项目成果

Genome-wide identification of zero nucleotide recursive splicing in Drosophila.

果蝇零核苷酸递归剪接的全基因组鉴定。

• 发表时间: 2015-05-21
• 影响因子: 64.8
• 作者: Duff, Michael O;Olson, Sara;Wei, Xintao;Garrett, Sandra C;Osman, Ahmad;Bolisetty, Mohan;Plocik, Ale;Celniker, Susan E;Graveley, Brenton R
• 通讯作者: Graveley, Brenton R