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

基本信息

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
项目类别：
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&apos 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

项目摘要

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（由应用提供）：长期非编码RNA（LNCRNA）最近出现为基因表达的调节剂，通过涉及染色质修饰，转录和转录后处理的机制运行。 LNCRNA超过数量蛋白编码基因，由相同的细胞机械转录，并且具有与信使RNA相似的结构特征。最近的研究表明，LNCRNA表达的正确编排对于正常的中枢神经系统发育是必要的，并且在几种人类神经系统疾病的病因中已经实施了它们的失调。与LNCRNA表达分析的进步相反，对其功能的了解少得多。了解LNCRNA功能需要开发能够鉴定其基因组靶标的技术的机制。一种这样的技术是RNA反义纯化（RAP）此I阶段SBIR提案旨在建立RAP在神经生物学模型系统中的鲁棒性，并评估其对LNCRNA的共同鉴定的潜力，并使用相关的DNA来识别基因组结合模式。直到最近开发的RAP才用于介绍培养细胞系中少数LNCRNA的基因组靶标。该阶段I SBIR提案旨在在神经生物学模型系统中建立RAP的鲁棒性，并评估其与LNCRNA相关蛋白的共同鉴定的潜力。 AIM 2努力将确定将RAP应用于人和小鼠神经元细胞系以及灌注小鼠脑的可行性。已知在大脑中已知表达的三个LNCRNA的DNA靶标将通过下一代测序确定。为了确定RAP是否可以通过质谱法来隔离和鉴定与LNCRNA相关的蛋白，LNCRNA HOTAIR已知与Polycomb抑制性复合物2（PRC2）相互作用，将用于概念证明。 PRC2是一种染色质修饰，含有EZH2和SUZ12蛋白。 AIM 3的目的是调整RAP，以成功识别Prc2成分蛋白EZH2或Suz12 Pepperides在Hotair中，其中包含用RAP分离的复合物。这些目标的成功完成将构成未来II期努力的基础，在这些阶段，在神经系统系统中使用RAP的潜力将进一步发展为一系列启用工具（产品和服务），这些工具（产品和服务）将加速LNCRNA在神经行为和疾病的病因中的功能分析。设想的产品包括LNCRNA探针集的自定义合成，RAP分析套件包含细节方案和试剂，以及提供LNCRNA相关蛋白或靶向基因组区域的分析作为服务。