Studies of Fragile X Syndrome (FXS): A search for therapeutics
Studies of Fragile X Syndrome (FXS): A search for therapeutics
Fragile X Syndrome (FXS) is due to a deficiency in the ubiquitously expressed RNA-binding protein FMRP. Our foray into FXS began serendipitously with the finding that FMRP co-immunoprecipitates with the nonsense-mediated RNA decay (NMD)-activated form of the key NMD factor, UPF1, in an RNase I-resistant manner.
NMD is Hyperactivated in FXS
Loss of fragile X protein, FMRP, is a leading single-gene cause of intellectual disability and autism, but the underlying mechanism remains poorly understood. We have found that FMRP deficiency results in hyperactivated NMD in FXS patient fibroblast-derived induced pluripotent stem cells (iPSCs). The underlying mechanism involves UPF1 recruiting and stabilizing FMRP on NMD-target mRNAs. Several lines of evidence indicate that FMRP acts as an NMD repressor: in the absence of FMRP, NMD targets are relieved from FMRP-mediated translational repression so that their half-lives are decreased and, for those NMD targets encoding NMD factors, increased translation produces abnormally high factor levels despite their hyperactivated NMD.
Data show that transcriptome-wide alterations caused by NMD hyperactivation have a role in the FXS phenotype. In support of this, small molecule-mediated inhibition of hyperactivated NMD that typifies FXS patient-derived iPSCs restores a number of neurodifferentiation markers, including those not deriving from NMD targets. Our mechanistic studies reveal that many molecular abnormalities in FMRP-deficient cells are attributable, either directly or indirectly, to misregulated NMD, offering new and completely unexpected insight into the molecular abnormalities of this incurable disease.
Inhibiting NMD as a Therapeutic for FXS
Our unexpected finding that the key NMD factor UPF1 interacts directly with FMRP, which when absent causes FXS, has led us to use stem-cell biology to better understand the consequences of the resulting misregulated NMD and how small molecule inhibitors of NMD might be used as a therapeutic.
Because it is becoming clear, even for genetic diseases that are detectable phenotypically only after birth, that abnormalities in gene expression begin to manifest in utero, we extended our studies to the Fmr1-knockout mouse, which is a commonly used experimental animal model for FXS. After defining NMD targets in mouse neuroblastoma cells, we have worked with URMC mouse developmental neurobiologist Chris Pröschel, PhD, and URMC surgeon Hitomi Sakano, MD, PhD to determine that NMD is hyperactivated in Fmr1-knockout mouse cortex, hippocampus, and cerebellum from embryonic day 16 to postnatal day 24.
Open Project: We aim to use this mouse to test the efficacy of small molecules that inhibit NMD in collaboration with biopharmaceutical companies and chemists, we aim to test the efficacy of small molecules that could serve as therapeutics (with funding from the FRAXA Research Foundation and the NYS Empire Discovery Institute) – in search of therapeutics for this incurable disease.
Identifying FMRP-binding sites and the mechanism by which FMRP represses mRNA translation
There has been considerable confusion in the literature over how FMRP binds RNA and which RNAs it binds. We combined RIP-seq, TRIC-seq, and SILAC coupled to LC-MS/MS to demonstrate that FMRP binds at GC-rich sequences of conservatively 400 mRNAs, only ~35% of which are NMD targets. Binding is most dense in 5'UTRs but also occurs within coding regions, independently of coding potential, and 3'UTRs, provided the mRNA harbors a poly(A) tail. FMRP binding to any of the three mRNA regions is sufficient to protect the mRNA from translation and deadenylation in a mechanism that depends on FMRP binding to poly(A)-bound PABPC1.
Open Project: Using human neuronal cells, we aim to study the functional significance of, and interactions among, those FMRP granule constituents we have identified, along with their post-translational modifications. Our goal is to understand the conversion of FMRP-bound mRNAs from a translationally inactive physically sequestered state to a translationally active state that is subjected to rapid deadenylation. We are also interested in how FMRP-bound mRNP is transported in polarized cells.