A novel method for identifying microRNA targets

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DESCRIPTION (provided by applicant): Abnormalities in microRNA signaling have been associated with multiple neurological and psychiatric diseases. Understanding these associations cannot proceed further, however, without better methods for determining microRNA targets. Because of the incomplete complementarity of microRNAs and their targets, predicting authentic microRNA:mRNA interactions can be difficult. Predictions typically rely on bioinformatics, but it is well known that the concurrence of different algorithms is distressingly low. Thus, empirical approaches for characterizing microRNA:mRNA interactions have received increasing attention. We have developed an approach that utilizes an epitope-tagged, dominant negative version of GW182, a component of the RNA-induced silencing complex (RISC), to purify microRNA:mRNA complexes prior to microRNA-mediated mRNA degradation. This method, which we term RISC-Trap, is considerably more robust than previously reported approaches and allows us to address fundamental problems in microRNA biology, such as, what proportion of mRNA targets undergo degradation versus translational arrest, which targets are direct or indirect, and whether some interactions occur specifically in neural versus non-neural cells. To begin to address these questions, we will use RNASeq to examine complexes containing miR-124, an abundant, neural-specific microRNA with a large data set of previously characterized targets. Our approach should be generally applicable to other systems, however, and should significantly increase understanding of the contributions of microRNAs to neurodevelopmental processes, plasticity, and certain neurological and psychiatric diseases. Our first specific aim is to use the RISC-trap approach to determine which miR-124 targets in HEK293 cells are regulated by mRNA degradation versus translational arrest. These studies will provide a comprehensive picture of the regulatory effects of an important brain microRNA and a straightforward and easily applicable method for identifying microRNA targets in general. Our second aim will be to identify miR-124 targets specific to neuronal cells and determine whether the mode of microRNA action differs. Although many important miR-124 targets are expressed in both neural and non-neural cells, specific neuronal targets are likely to exist as well. We will identify these neural-specific targets by infecting SH-SY5Y neuroblastomas and primary hippocampal neurons with a dnGW182 lentivirus and analyzing the targets as described above. Some targets will be detected only in neuronal cells because their expression is linked to this cell type. Neural-specific microRNA interactions involving mRNAs expressed in both cell types may indicate the involvement of essential RNA binding proteins. We believe that the RISC-trap assay may uncover new aspects of microRNA function.