PROJECT SUMMARY: Fragile X Syndrome (FXS) is the most common form of both inherited intellectual disability and monogenic cause of autism. It is caused by the loss of Fragile X Messenger Ribonucleoprotein 1 (FMR1/FMRP) protein. FMRP, an RNA-binding protein, regulates target mRNAs through a variety of modes, such as controlling mRNA localization, stabilization, editing, and/or translation. Translational repression by FMRP is especially important in neuronal processes, where protein synthesis must be controlled in an orderly fashion. In the absence of FMRP, translational control is disrupted resulting in the over-production of dendritic proteins contributing to the FXS neuron phenotype of immature spines and hyperexcitability. Proper translation regulation by FMRP requires microRNAs (miRNAs) and the RNA-induced silencing complex {RISC). The RISC regulates gene expression post-transcriptionally by utilizing base matching between a miRNA and target mRNA, which allows for specific recognition of target mRNAs. It has been shown that miRNAs are dysregulated in several FXS models; however, no study has unbiasedly characterized the complete mi RNA landscape in human FXS models. Another unaddressed aspect of FMRP-miRNA mediated gene regulation is whether the interactions are always cooperative. Recent studies have highlighted an underappreciated role for FMRP in enhancing translation rather than suppressing it. Thus, I hypothesize that FMRP loss alters the miRNA landscape, and that FMRP not only coordinates miRNA-mediated repression, but in some cases antagonizes the miRNA-RISC machinery to protect transcripts from improper regulation. To test my hypotheses, I will conduct experiments aimed at the following goals: 1.) Test whether loss of FMRP alters global neuron miRNA expression patterns, miRNA incorporation into the RISC, and localization of miRNAs to neuronal processes. 2.) Determine whether FMRP-microRNA interactions regulate global and/or local translation of FMRP target mRNAs. To accomplish these aims, I will require new training in induced-pluripotent stem cell cultures, neural organoids, and microscopy. My sponsor, Dr. Gary Bassell, has expertise in these methods, and the scientific community of Emory University School of Medicine will offer me an excellent training environment. Successful completion of this fellowship proposal will not only reveal the extent to which miRNAs are dysregulated in human models of FXS, but also reveal whether FMRP and miRNAs always act in a unimodal manner. Of equal importance, this project coupled with Dr. Bassell's mentorship will allow me to transition from a molecular cancer biologist to an independent molecular and cellular neurobiologist.