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

描述（由申请人提供）：用于细胞特异性鉴定RNA结合蛋白靶点的新工具。这个提议源于我对果蝇昼夜节律系统转录后调控的兴趣。尽管历史上关注的焦点一直是转录调控，但现在人们对所有发生在控制昼夜运动活动节律的中枢脑神经元中的转录后调控产生了浓厚的兴趣。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，包括关键的起搏器细胞。我们有遗传和生物化学证据表明，RNA结合蛋白（RBP） Hrp48对这些细胞内的昼夜节律保持做出了重大贡献。最重要的问题是，Hrp48的RNA靶标是什么。然而，鉴定体内特异性rbp的mRNA靶点具有挑战性，特别是在少量离散神经元中。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-蛋白鉴定的金标准可以说是HITS-CLIP（高通量测序-交联和免疫沉淀），即在体内用紫外线将蛋白质与RNA交联，然后进行免疫沉淀，RNAse酶切，并对与该蛋白质交联的RNA片段进行深度测序。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，对于我们的目的来说，不太可能有足够的材料

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.