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), which is an RNA binding protein involved in the transport and translation regulation of specific messenger RNA (mRNA) targets. We have determined that FMRP has nuclease activity, being able to process precursor microRNAs (pre-miRNAs), potentially being involved in the mature miRNA biogenesis. The parental 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. Under this aim we will determine: (i) if the FMRP nuclease activity resides in one of his KH domains and identify the active site residues responsible for this activity by point mutations; (ii) if FMRP S500D has higher nuclease activity as compared with unphosphorylated FMRP due different dimerization properties and (iii) if the 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. Under this specific aim we will: (i) determine if FMRP and FMRP S500D cleave pre-miRNAs into mature miRNAs; (ii) determine if they have additional substrates such as RNA perfect duplex, RNA single strand, RNA G quadruplex, DNA-RNA hybrid duplex, DNA duplex; (iii) characterize the kinetics of their nuclease activity. Similar experiments will pe performed to test the nuclease activity of the FMRP paralogs FXR1P and FXR2P. 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. Under this aim we will investigate if FMRP and FMRP S500D, as well as FXR1P and FXR2P interact with the SARS-CoV-2 3’-UTR genome, cleaving conserved stem- loops either to potentially yield viral miRNAs or functioning in the antiviral host response. Fluorescence spectroscopy will be used in all three specific aims of the parent R15 proposal, with the requested Fluorolog-QM-75-11-C spectrofluorometer and accessories being essential for the success of the proposed experiments.