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.

PUBLIC HEALTH RELEVANCE: A new tool for the cell-specific identification of RNA binding protein targets. This application proposes to develop a sensitive new approach to the identification of RNA binding protein (RBP) substrates. It involves creation of a fusion protein between an RBP and the catalytic domain of a RNA editing enzyme like ADAR (adenosine deaminase). The fusion protein will have removed the RNA targeting features of ADAR, so the catalytic domain will be delivered at high local concentration to RNA targets of the RBP. To improve sensitivity, RNA can be sequenced from the cells of interest, either after IP or after neuronal purification. Preliminary data from tissue culture experiments indicate that the approach is promising and warrants further development, both in culture and in fly neurons, as it will provide a novel and widely applicable new technique for the cell-specific identification of RBP targets.