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)：RNA结合蛋白靶标的细胞特异性鉴定的新工具。这项提议源于我对果蝇昼夜节律系统转录后调控的兴趣。虽然历史上的焦点一直是转录调控，但现在人们对所有发生在中央大脑神经元内的转录后调控非常感兴趣，这些神经元控制着昼夜运动活动的节奏。特别令人感兴趣的是成人大脑两侧的8-9个神经元，它们表达神经肽PDF，包括关键的起搏细胞。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律起着重要作用。那么首要的问题就变成了，Hrp48的RNA靶标是什么。然而，在体内识别特定限制性商业惯例的mRNA靶标是具有挑战性的，特别是在少数离散神经元中。这一挑战绝不局限于Hrp48，还延伸到许多其他重要的RNA结合蛋白，如dFMRP(脆性X智力低下蛋白)。鉴定RNA-蛋白质的金标准可以说是HITS-CLIP(高通量测序-交联和免疫沉淀)，即在体内用紫外光将蛋白质与RNA交联，然后进行免疫沉淀、核糖核酸酶消化和与该蛋白质交联的RNA片段的深度测序。然而，这种方法并不完美：例如，UV交联的效率是可变的，通常很低(1-5%)。更重要的是，就我们的目的而言，不太可能有足够的材料来 从离散的脑神经元群体中提取标记蛋白的纯度以用于成功鉴定 靶向mRNAs。这项建议旨在通过开发一种完全不同的方法来识别RBP底物来规避这些问题。它包括在RBP和像ADAR(腺苷脱氨酶)这样的RNA编辑酶的催化域之间创建融合蛋白。这种酶将腺苷脱氨为肌苷，核糖体机构将肌苷解释为鸟苷。ADAR编辑的底物可以通过测序RNA来鉴定，即存在相当大比例的G，其中通常只有基因编码的A。融合蛋白将消除ADAR的RNA识别特征，因此催化结构域以高局部浓度递送到RBP的RNA靶标。如果有必要，为了提高灵敏度，可以对感兴趣的细胞进行RNA测序，无论是在IP之后，还是在神经元纯化之后。细胞培养实验的初步数据表明，该方法在组织培养和苍蝇神经元中都是有前景的，值得进一步发展，因为它将为RBP靶点的细胞特异性识别提供一种新的、广泛适用的新技术。