A new tool for the cell-specific identification of RNA binding protein targets

用于细胞特异性鉴定 RNA 结合蛋白靶标的新工具

• 批准号: 8640299
• 负责人: MICHAEL ROSBASH
• 金额: $ 28.12万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8640299
• 关键词: Adenosine; Adult; Biochemical; Biological Assay; Biology; Brain; Catalytic Domain; Cell Culture Techniques; Cells; Chimeric Proteins; Circadian Rhythms; Computer-Assisted Image Analysis; Data; Development; Digestion; Drosophila genus; Enzymes; Fragile X Mental Retardation Protein; Genetic; Gold; Guanosine; Immunoprecipitation; Inosine; Messenger RNA; Methods; Motor Activity; Neurons; Neuropeptides; Neurosciences; Population; Post-Transcriptional Regulation; Proteins; RNA; RNA Editing; RNA Sequences; RNA-Binding Proteins; Side; System; Techniques; Transcriptional Regulation; Variant; adenosine deaminase; crosslink; deep sequencing; design; fly; improved; in vivo; interest; link protein; nodal myocyte; novel; novel strategies; public health relevance; research study; tissue culture; tool

DESCRIPTION (provided by applicant): A new tool for the cell-specific identification of RNA binding protein targets. This proposal has its origins in my interest in post-transcriptional regulation in the Drosophila circadian system. Although historically the focus has been on transcriptional regulation, there is now substantial interest in all of the post-transcriptional regulation that occurs within the central brain neurons that govern circadian locomotor activity rhythms. Of particular interest are 8-9 neurons on each side of the adult brain, which express the neuropeptide PDF and include key pacemaker cells. We have genetic and biochemical evidence that the RNA binding protein (RBP) Hrp48 makes a substantial contribution to circadian timekeeping within these cells. The overarching question then becomes, what are the RNA targets of Hrp48. However, the identification of in vivo mRNA targets of specific RBPs is challenging, especially within small numbers of discrete neurons. This challenge is by no means limited to Hrp48 and extends to many other important RNA-binding proteins like dFMRP (Fragile X Mental Retardation Protein). The gold standard for RNA-protein identification is arguably HITS-CLIP (High Throughput Sequencing- Cross Linking and Immunoprecipitation), i.e., in vivo cross-linking of protein to RNA with UV followed by immunoprecipitation, RNAse digestion, and deep-sequencing of RNA fragments cross-linked to that protein . However, the method is imperfect: for example, the efficiency of UV cross-linking is variable, and typically very low (1-5%). More importantly for our purposes, there is unlikely to be sufficient material of a sufficient purity from a tagged protein in discrete populations of brain neurons for successful identification of target mRNAs. This proposal is designed to circumvent these issues by developing an entirely different approach to the identification of RBP substrates. It involves creating a fusion protein between an RBP and the catalytic domain of a RNA editing enzyme like ADAR (adenosine deaminase). This enzyme deaminates adenosine to inosine, which is interpreted by the ribosomal machinery as a guanosine. ADAR edited substrates can be identified by sequencing RNA, i.e., the presence of a substantial percentage of G where there is normally only a genomically-encoded A. The fusion protein will have removed the RNA recognition features of ADAR, so the catalytic domain is delivered at high local concentration to RNA targets of the RBP. To improve sensitivity if necessary, RNA can be sequenced from the cells of interest, either after IP or after neuronal purification. Preliminary data from cell culture experiments indicate that the approach is promising and warrants further development, both in tissue culture and in fly neurons, as it will provide a novel and widely applicable new technique for the cell-specific identification of RBP targets.

描述（由申请人提供）：一种用于细胞特异性鉴定RNA结合蛋白靶标的新工具。该提议起源于我对果蝇昼夜节律系统转录后调节的兴趣。尽管从历史上看，重点一直放在转录调节上，但现在在控制昼夜节律运动节奏的中央脑神经元内发生的所有转录后调节都有重大的兴趣。特别令人感兴趣的是成年大脑两侧的8-9个神经元，它们表达神经肽PDF并包括关键的起搏器细胞。我们有遗传和生化证据表明，RNA结合蛋白（RBP）HRP48对这些细胞内的昼夜节时间做出了重大贡献。然后，总体问题变成了HRP48的RNA靶标。但是，特定RBP的体内mRNA靶标的鉴定是具有挑战性的，尤其是在少数离散神经元中。这一挑战绝不限于HRP48，并且扩展到许多其他重要的RNA结合蛋白（如DFMRP）（脆弱的X智力低下蛋白）。 RNA-蛋白识别的金标准可以说是搅拌-CLIP（高吞吐量测序 - 交叉链接和免疫沉淀），即用紫外线与紫外线进行蛋白质与RNA的体内交联，然后进行免疫沉淀，RNase消化，深层rna rna fragments交叉链接到该蛋白质。但是，该方法不完美：例如，紫外线交联的效率是可变的，通常非常低（1-5％）。更重要的是出于我们的目的，不太可能有足够的材料 在脑神经元离散群体中的标记蛋白质纯度以成功识别 目标mRNA。该建议旨在通过开发完全不同的RBP底物的方法来避免这些问题。它涉及在RBP和RNA编辑酶（如ADAR）（腺苷脱氨酶）等RNA编辑酶的催化结构域之间创建融合蛋白。这种酶将腺苷脱离至肌苷，核糖体机械将其解释为鸟嘌呤。 ADAR编辑的底物可以通过测序RNA来识别，即，在通常只有基因组编码的A。融合蛋白将消除ADAR的RNA识别特征的G，因此催化域以高局部浓度输送到RNA的RNA靶标。为了提高敏感性，可以在IP之后或神经元纯化后从感兴趣的细胞中对RNA进行测序。来自细胞培养实验的初步数据表明，该方法是有希望的，并需要在组织培养和蝇神经元中进一步发展，因为它将为RBP靶标的细胞特异性鉴定提供一种新颖且广泛适用的新技术。