Neurobiology toolbox for identification of lncRNA targets and associated proteins

用于识别 lncRNA 靶标和相关蛋白的神经生物学工具箱

• 批准号: 8902690
• 负责人: Theresa K. Kelly
• 金额: $ 22.42万
• 依托单位: ACTIVE MOTIF, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8902690
• 关键词: Acceleration; Adult; Alzheimer' s Disease; Anti-Sense Probes; Antisense RNA; Archives; Behavior; Binding; Biological; Biological Assay; Biological Models; Biology; Boxing; Brain; Brain region; Breast Cancer Cell; Cell Line; Cells; Chromatin; Code; Complex; Custom; DNA; DNA Methylation; Development; Digestion; Disease; EZH2 gene; Ensure; Etiology; Feasibility Studies; Fibroblasts; Future; Gene Expression; Generations; Genes; Genetic Transcription; Genomic Segment; Genomics; Glioma; Goals; Histones; Homeostasis; Housing; Human; Huntington Disease; Length; Lung; Mass Spectrum Analysis; Messenger RNA; Methodology; Methods; Molecular Profiling; Mus; Neuraxis; Neurobiology; Neuroblastoma; Neurodegenerative Disorders; Neurologic; Neurons; Pattern; Peptides; Phase; Polycomb; Post-Translational Protein Processing; Preparation; Process; Proteins; Protocols documentation; Publishing; RNA; Reagent; Regulator Genes; Reverse Transcription; Sampling; Scientist; Sequence Analysis; Services; Small Business Innovation Research Grant; Source; Specificity; System; Techniques; Technology; Testing; Tissues; Untranslated RNA; base; brain tissue; cell type; chromatin modification; crosslink; design; genome-wide; improved; insight; nervous system development; nervous system disorder; neuropathology; next generation sequencing; public health relevance; success; technique development; tool

 DESCRIPTION (provided by applicant): Long non-coding RNAs (lncRNAs) have recently emerged as regulators of gene expression, functioning via mechanisms that involve chromatin modification, transcription and post-transcriptional processing. LncRNAs exceed the number protein coding genes, are transcribed by the same cellular machinery, and have similar structural features as messenger RNAs. Recent studies have shown that correct orchestration of lncRNA expression is necessary for normal central nervous system development and their dysregulation has been implicated in the etiology of several human neurological diseases. In contrast to advances in lncRNA expression profiling, much less is known about their function. Gaining insight into the mechanisms by which lncRNAs function requires the development of techniques that enable the identification of their genomic targets. One such technique is RNA antisense purification (RAP) which uses a pool of overlapping antisense biotinylated probes to capture lncRNAs and the associated DNA is used to identify the genomic binding patterns. Developed only recently, RAP has been use to profile the genomic targets of a handful of lncRNAs in cultured cell lines. This Phase I SBIR proposal intends to establish the robustness of RAP in neurobiological model systems and to assess its potential for the co-incident identification of lncRNA-associated proteins. This would enable scientists to directly identify which lncRNAs and proteins are both present at a given genomic locus using the same technique. Aim 1 efforts will establish transfer of technical know-how of using RAP to target three lncRNAs whose genomic distribution has been determined in specific cell lines. Aim 2 efforts will establish the feasibility of applying RAP to human and mouse neural cell lines and to perfused mouse brain. The DNA targets of three lncRNAs known to be expressed in brain will be determined by next generation sequencing. To establish whether RAP can be used to isolate and identify lncRNA-associated proteins by mass spectrometry, the lncRNA HOTAIR, which is known to interact with the Polycomb Repressive Complex 2 (PRC2), will be used for proof of concept. PRC2 is a chromatin modifying complex containing the EZH2 and SUZ12 proteins. The goal of Aim 3 is to adapt RAP to enable the successful identification of PRC2 constituent proteins EZH2 or SUZ12 peptides in HOTAIR containing complexes isolated by RAP. Successful completion of these objectives will form the basis of future Phase II efforts where the potential of utilizing RAP in neurological systems will be further developed into a suite of enabling tools (products and services) that will accelerate the functional analysis of lncRNAs in the etiology of neurological behaviors and diseases. The products envisioned include custom synthesis of lncRNA probe sets, RAP assay kits containing a detail protocol and reagents, and providing profiling of lncRNA associated proteins or targeted genomic regions as a service.

 描述（由申请人提供）：长非编码RNA（lncRNA）最近已经作为基因表达的调节剂出现，通过涉及染色质修饰、转录和转录后加工的机制发挥作用。LncRNA超过蛋白质编码基因的数量，由相同的细胞机制转录，并具有与信使RNA相似的结构特征。最近的研究表明，lncRNA表达的正确协调对于中枢神经系统的正常发育是必要的，并且它们的失调与几种人类神经系统疾病的病因有关。与lncRNA表达谱的进展相反，对其功能的了解要少得多。深入了解lncRNA的功能机制需要开发能够识别其基因组靶点的技术。一种这样的技术是RNA反义纯化（RAP），其使用重叠的反义生物素化探针池来捕获lncRNA，并且相关的DNA用于鉴定基因组结合模式。RAP是最近才发展起来的，它已被用于分析培养细胞系中少数lncRNA的基因组靶点。该I期SBIR提案旨在建立RAP在神经生物学模型系统中的稳健性，并评估其用于同时鉴定lncRNA相关蛋白的潜力。这将使科学家能够使用相同的技术直接识别哪些lncRNA和蛋白质都存在于给定的基因组位点。目标1的努力将建立使用RAP靶向三种lncRNA的技术诀窍的转让，这些lncRNA的基因组分布已经在特定的细胞系中确定。目的2：建立RAP应用于人类和小鼠神经细胞系以及灌流小鼠脑的可行性。已知在脑中表达的三种lncRNA的DNA靶标将通过下一代测序来确定。为了确定RAP是否可用于通过质谱法分离和鉴定lncRNA相关蛋白，将使用已知与Polycomb抑制复合物2（PRC2）相互作用的lncRNA HOTAIR进行概念验证。PRC2是含有EZH2和SUZ12蛋白的染色质修饰复合物。目的3的目标是使RAP适应于能够成功鉴定通过RAP分离的含有HOTAIR的复合物中的PRC2组成蛋白EZH2或SUZ12肽。这些目标的成功完成将成为未来第二阶段工作的基础，其中在神经系统中利用RAP的潜力将进一步发展成为一套使能工具（产品和服务），这将加速lncRNA在神经行为和疾病病因学中的功能分析。设想的产品包括lncRNA探针组的定制合成、包含详细方案和试剂的RAP测定试剂盒，以及提供lncRNA相关蛋白或靶向基因组区域的分析作为服务。