Sequence-specific Capture for Discovering Protein-IncRNA Interactions in Prostate Cancer

用于发现前列腺癌中蛋白质-IncRNA 相互作用的序列特异性捕获

• 批准号: 8857740
• 负责人: DAVID F. JARRARD
• 金额: $ 34.42万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-04 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8857740
• 关键词: Address; Affect; Antibodies; Beds; Benchmarking; Binding; Binding Proteins; Biological; Biological Markers; Biology; Cell Culture Techniques; Cell Line; Cells; Chromatin; Complex; DNA; Development; Disease; Disease Marker; Disease Progression; Elements; Epithelial Cells; Epithelium; Evaluation; Formaldehyde; Gene Expression; Gene Expression Regulation; Genetic Transcription; Genome; Human; Human Cell Line; Immunoprecipitation; Individual; Indolent; Investigation; Knowledge; LNCaP; Learning; Location; Malignant Neoplasms; Malignant neoplasm of prostate; Mass Spectrum Analysis; Messenger RNA; Methodology; Methods; Molecular; Normal tissue morphology; Nucleic Acid Hybridization; Nucleic Acids; Onset of illness; PRC1 Protein; Patients; Pattern; Phenotype; Play; Procedures; Process; Prostate; Prostatic Neoplasms; Protein Binding; Protein Databases; Proteins; Proteomics; RNA; RNA-Protein Interaction; Reagent; Recombinant DNA; Role; Sampling; Scientist; Set protein; Technology; Testing; Time; Tissue Sample; Tissues; Transcriptional Regulation; Translational Regulation; Tumor Tissue; Untranslated RNA; Validation; Work; Yeasts; aptamer; crosslink; differential expression; histone modification; in vivo; innovation; insight; interest; new technology; novel marker; novel strategies; promoter; prostate cancer cell line; protein complex; protein crosslink; public health relevance; targeted sequencing; technology validation; tool; transcriptome sequencing

 DESCRIPTION (provided by applicant): Protein-nucleic acid interactions play key roles in both transcriptional and translational regulation, not only in the direct interaction of proteins with DA and mRNA to regulate these processes, but also the interactions among proteins, lncRNAs, and DNAs. Understanding these interactions requires knowledge of the molecules that are involved. lncRNAs interact with proteins to achieve several consequences in gene regulation. First, lncRNAs can act as a "molecular sink" for proteins that may interact with DNA or RNA. Their interaction with the lncRNA thus diverts the protein from binding to its primary target. lncRNAs can also act as guides for proteins, leading them to their DNA targets to either repress or activate transcription. Finally, lncRNAs can act as platforms upon which molecules can congregate to perform a function, such as histone modification, as a team at a specific location and time. Each of these functions requires the interaction of lncRNAs with a diverse set of proteins. Knowledge of the proteins bound to a specific lncRNA will allow for determination of the mechanisms by which that lncRNA affects gene expression. Unraveling this tremendous diversity of interactions demands tools capable of rapid identification and quantification of proteins. In this proposal we focus on the particular problem of identifying the proteins that are bound to specific lncRNA molecules, their relationship to the progression of prostate cancer and their potential as markers of the disease and its progression. We describe an RNA-centric approach to discovery of the lncRNA-binding proteins that is innovative compared with existing technologies in two major ways. First, crosslinking of proteins to lncRNAs is done in vivo under normal cellular conditions. Therefore, proteins and lncRNAs will be folded normally, will be present at their normal cellular concentrations, and will be in their normal cellular locations. Second, sequence-specific capture of the lncRNA is used to extract the RNA of interest along with its crosslinked proteins. This is a universal capture strategy that does not rely on the availability of any other capture reagent (e.g. antibody or aptamer). Following enrichment by sequence-specific capture, mass spectrometry will be utilized to identify and quantify the associated proteins.

 描述（由申请人提供）：蛋白质-核酸相互作用在转录和翻译调节中起关键作用，不仅在蛋白质与DA和mRNA的直接相互作用中调节这些过程，而且在蛋白质、lncRNA和DNA之间的相互作用中。理解这些相互作用需要了解所涉及的分子。lncRNA与蛋白质相互作用以在基因调控中实现若干结果。首先，lncRNA可以作为蛋白质的“分子库”，这些蛋白质可以与DNA或RNA相互作用。因此，它们与lncRNA的相互作用使蛋白质从与其主要靶标的结合转移。lncRNA还可以作为蛋白质的向导，引导它们到达其DNA靶点以抑制或激活转录。最后，lncRNA可以作为平台，分子可以聚集在其上执行功能，例如组蛋白修饰，作为一个团队在特定的位置和时间。这些功能中的每一个都需要lncRNA与一组不同的蛋白质相互作用。与特定lncRNA结合的蛋白质的知识将允许确定lncRNA影响基因表达的机制。解开这种巨大的相互作用的多样性需要能够快速识别和定量蛋白质的工具。 在这个建议中，我们专注于识别蛋白质的特殊问题， 与特定lncRNA分子结合，它们与前列腺癌进展的关系以及它们作为疾病及其进展的标志物的潜力。我们描述了一种以RNA为中心的方法来发现lncRNA结合蛋白，与现有技术相比，该方法在两个主要方面具有创新性。首先，蛋白质与lncRNA的交联在正常细胞条件下在体内进行。因此，蛋白质和lncRNA将正常折叠，将以其正常细胞浓度存在，并且将处于其正常细胞位置。其次，lncRNA的序列特异性捕获用于提取感兴趣的RNA连同其交联蛋白质沿着。这是一种通用捕获策略，不依赖于任何其他捕获试剂（例如抗体或适体）的可用性。通过序列特异性捕获富集后，将使用质谱法鉴定和定量相关蛋白质。