Amongst the most intriguing and complex questions in neuroscience is that concerning the molecular mechanisms by which synapses participate in learning and memory. Beginning as early as the 1960s, a growing body of evidence indicates that new protein synthesis is required for learning and long-term memory formation.1 Additionally, it has been shown that activity-dependent protein synthesis is highly specific, as only a small subset of available transcripts are translated following neuronal activity.2,3 There is also evidence that local protein synthesis at synapses mediates plasticity and that perturbations in translation are associated with disorders of cognitive function such as autism.4,5 The realization of the proximity of protein synthesis to the sites of synaptic transmission, the specificity of transcripts that are translated, and its disruption in disease has underscored the importance of understanding the nature of post-transcriptional regulatory mechanisms that shape the complement of proteins in neurons and at synapses. Since their discovery in 1993, microRNAs (miRNAs) have been appreciated for their breadth of function as post-transcriptional regulators of protein synthesis by translation inhibition and transcript destabilization.6 miRNAs regulate neuronal plasticity and dendritic spine morphogenesis, are implicated in higher-order brain functions such as memory and cognitive dysfunction, and proteins involved in miRNA biogenesis and function are found in neurons, including near synapses.1 The evolutionarily conserved let-7 family of miRNAs has emerged as a critical mediator of post-transcriptional gene regulation in many growth-related processes including developmental timing (C. elegans7 and D. melanogaster8,9), body axis programming (M. musculus10), metabolism (M. musculus11), and cancer (M. musculus12 and H. sapiens13). The let-7 family of miRNAs are highly abundant in mature differentiated neurons and work from our lab and others has shown that let-7 miRNA levels can be regulated by neuronal activity3,14-16 and are disrupted in a mouse model lacking the fragile X mental retardation protein (FMRP).31 However, an approach to unambiguously determine the genome-wide identity of mRNA targets for let-7 and other miRNAs has not been possible until recently. This project employs a modified version of the recently developed CLEAR-CLIP technique17 that involves cross-linking and immunoprecipitation followed by intermolecular ligation of endogenous RNAs bound to Argonaute and high-throughput sequencing (CIMERA- seq). CIMERA-seq will allow for critical miRNA targets and miRNA-regulatory mechanisms governing protein synthesis in the mammalian CNS to be explored in great detail. The hypothesis of this proposal is that lowered let-7 miRNA levels observed in vitro and in vivo in the FMRP-deficient brain will produce changes in the miRNA- target profile consistent with altered neuronal plasticity, synapse overgrowth, and protein synthesis observ...