Biochemical characterization of a novel Fragile X Mental Retardation Protein nuclease function

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Project Summary Fragile X mental retardation syndrome is the most common form of inherited mental impairement, affecting ~ 1 in 4000 males and ~ 1 in 6000 females. The syndrome is caused by the loss of a normal cellular protein, named the fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein involved in the transport and translation regulation of specific messenger RNA (mRNA) targets. The mechanisms by which FMRP exerts its translation regulator function are not known, however it has been proposed that the protein works in conjunction with the microRNA (miRNA) pathway to regulate local protein synthesis in response to synaptic input. We have determined that FMRP has nuclease activity, being able to process precursor microRNAs (pre-miRNAs), potentially being involved in the mature miRNA biogenesis. This proposal, which will characterize this novel FMRP function, has the following specific aims: AIM I. Identification of the FMRP domain(s) responsible for its nuclease activity. We hypothesized that the FMRP nuclease activity resides in one of his K homology domains and to test this hypothesis we will produce several FMRP constructs lacking one or more of these domains. We determined that phosphorylated FMRP has higher nuclease efficiency than the unphosphorylated FMRP and we will test if this is caused by their different dimerization properties. Finally, we will also test if the FMRP paralogs, FXR1P and FXR2P, which share with FMRP the KH0, KH1 and KH2 domains, also have nuclease activity. AIM II. Biochemical characterization of the FMRP nuclease activity. We will determine if the pre-miRNA FMRP and its phosphorylated mimic can cleave pre-miRNAs into mature miRNAs. Additionally, we will determine if FMRP has additional substrates for its nuclease activity such as RNA perfect duplex, RNA single strand, RNA G quadruplex, DNA-RNA hybrid duplex, DNA duplex. Finally, we will characterize the kinetics of the FMRP nuclease. AIM III. Investigation of the FMRP interactions with the SARS-CoV-2 3’-UTR genome and of the potential role played by its nuclease activity in this viral system. Similar to its demonstrated role of restrictive factor in ZIKA virus infection, it has been proposed that FMRP might play a role in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the virus responsible for the current COVID-19 pandemic. We will test if FMRP cleaves various stem-loops within the SARS-CoV-2 RNA genomic 3’-untranslated region using its nuclease function either to potentially yield viral miRNAs or functioning in the antiviral host response.