Understanding the Genome Maintenance Function of the Fragile X Protein (FMRP)

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PROJECT SUMMARY/ABSTRACT The main goal of the proposed research project is to understand the nuclear functions of FMRP and their impact on the etiological basis for the Fragile X syndrome (FXS). FXS occurs when mutations in the FMR1 gene cause the absence or loss of function of FMRP. FMRP has been primarily characterized as a translation repressor of a wide range of mRNA substrates in the cytoplasm, but its nuclear functions are not well understood. We recently reported that cells derived from FXS patients suffer genome-wide DNA double-strand breaks (DSBs) when under replication stress. Moreover, the DNA DSBs in FXS cells occurred near sequences that are prone to form DNA:RNA hybrids called R-loops during gene transcription. This finding suggested a new function of FMRP in preventing R-loop-induced DSBs during replication stress, thereby maintaining genome stability. Following this paradigm-shifting discovery, we found that FMRP directly binds R- loops and DHX9, an R-loop resolvase, through multivalent interactions. Therefore, our study provides a mechanism through which FMRP assists in R-loop resolution by bridging R-loops and R-loop resolvases on the chromatin. Additionally, we observed reduced gene expression in virtually all DNA repair pathways in the FXS genome, with and without replication stress, and linked this phenotype to an impaired p53 pathway. In this proposed study we will extend our analysis to ask if FMRP deficiency causes genome-wide DNA damage in human neurons. We will systematically identify and compare DNA DSBs and R-loops in neurons induced from FXS patient induced pluripotent cells and by doing so, we will discern those “at-risk” genes that are most susceptible to DSBs and down-regulation in cells lacking FMRP, thus providing a short list of potential therapeutic targets for FXS. In addition, we have observed direct interaction between FMRP and DHX9, an RNA:DNA hybrid helicase. We will determine the mechanism through which FMRP bridges R-loop and DHX9 to facilitate R-loop resolution. We will also probe the FMRP nuclear proteome in human neurons to identify additional factors that interact with FMRP on the chromatin. Our proposed project will further our understanding of the FMRP genome maintenance function and promises to shed new light into the etiological basis for FXS.