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: 8913110
• 负责人: MICHAEL ROSBASH
• 金额: $ 27.86万
• 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 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8913110
• 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; Health; High-Throughput Nucleotide Sequencing; 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; research study; tissue culture; tool; transcriptome sequencing

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。这个提议源于我对果蝇昼夜节律系统转录后调控的兴趣。尽管历史上焦点一直集中在转录调节上，但现在人们对控制昼夜运动活动节律的中枢大脑神经元内发生的所有转录后调节产生了浓厚的兴趣。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 并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做出了重大贡献。那么最重要的问题就变成了，Hrp48 的 RNA 靶标是什么。然而，识别特定 RBP 的体内 mRNA 靶点具有挑战性，特别是在少量离散神经元内。这一挑战绝不仅限于 Hrp48，还扩展到许多其他重要的 RNA 结合蛋白，如 dFMRP（脆性 X 智力迟钝蛋白）。 RNA-蛋白质鉴定的黄金标准可以说是 HITS-CLIP（高通量测序-交联和免疫沉淀），即利用 UV 在体内将蛋白质与 RNA 交联，然后进行免疫沉淀、RNAse 消化以及与该蛋白质交联的 RNA 片段的深度测序。然而，该方法并不完美：例如，UV 交联的效率是可变的，并且通常非常低（1-5%）。更重要的是，对于我们的目的来说，不太可能有足够的材料 离散脑神经元群体中标记蛋白的纯度，以便成功识别 目标 mRNA。该提案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。它涉及在 RBP 和 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。这种酶将腺苷脱氨基为肌苷，肌苷被核糖体机器解释为鸟苷。 ADAR 编辑的底物可以通过对 RNA 进行测序来识别，即存在相当大比例的 G，而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，因此催化结构域以高局部浓度递送至 RBP 的 RNA 靶标。为了在必要时提高灵敏度，可以在 IP 或神经元纯化后对感兴趣的细胞的 RNA 进行测序。细胞培养实验的初步数据表明，该方法在组织培养和果蝇神经元中都是有前景的，值得进一步开发，因为它将为 RBP 靶点的细胞特异性识别提供一种新颖且广泛适用的新技术。