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

描述（由申请人提供）：用于RNA结合蛋白靶标的细胞特异性鉴定的新工具。这个提议起源于我对果蝇昼夜节律系统中转录后调控的兴趣。虽然历史上的重点一直是转录调控，现在有大量的兴趣在所有的转录后调节，发生在中枢神经元内的昼夜运动活动节律。特别令人感兴趣的是成人大脑两侧各有8-9个神经元，它们表达神经肽PDF，包括关键的起搏细胞。我们有遗传和生物化学证据表明，RNA结合蛋白（RBP）Hrp 48对这些细胞内的昼夜节律计时做出了重大贡献。最重要的问题是，Hrp 48的RNA靶点是什么。然而，特定RBP的体内mRNA靶点的鉴定是具有挑战性的，特别是在少量离散神经元内。这一挑战绝不限于Hrp 48，并延伸到许多其他重要的RNA结合蛋白，如dFMRP（脆性X智力迟钝蛋白）。RNA-蛋白质鉴定的金标准可以说是HITS-CLIP（高重复测序-交联和免疫沉淀），即，用UV使蛋白质与RNA体内交联，随后免疫沉淀、RNA酶消化和与蛋白质交联RNA片段的深度测序。然而，该方法是不完美的：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，不太可能有足够的材料， 从离散的脑神经元群体中标记的蛋白质中获得纯度， 的靶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RNA编辑酶如阿达尔（腺苷脱氨酶）的催化结构域之间创建融合蛋白。这种酶将腺苷脱氨为肌苷，肌苷被核糖体机制解释为鸟苷。阿达尔编辑的底物可以通过测序RNA来鉴定，即，在通常只有基因组编码的A的地方存在相当大比例的G。融合蛋白将去除阿达尔的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。如果需要，为了提高灵敏度，可以在IP后或神经元纯化后从感兴趣的细胞中测序RNA。从细胞培养实验的初步数据表明，该方法是有前途的，并保证进一步发展，无论是在组织培养和苍蝇神经元，因为它将提供一种新的和广泛适用的新技术的细胞特异性识别的RBP目标。

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.