Dissecting the RNA-Binding Specificity and Role in Regulating mRNA Translation of the Fragile X Mental Retardation Protein

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and is caused by a deficiency of the fragile X mental retardation protein (FMRP) in neurons. FMRP regulates the translation of numerous mRNAs within dendritic synapses, but how FMRP recognizes these target mRNAs remains unknown. FMRP has KH0, KH1, KH2, and RGG domains, which are thought to bind to specific RNA recognition elements (RREs). Several studies used high-throughput methods to identify various RREs in mRNAs that FMRP may bind to in vivo. However, there is little overlap in the mRNA targets identified by each study. To determine the specificity of FMRP for the RREs, we performed quantitative in vitro RNA binding studies with various constructs of human FMRP. Our studies show that the KH domains do not bind to the previously identified RREs. To further investigate the RNA-binding specificity of FMRP, we developed a new method called Motif Identification by Analysis of Simple sequences (MIDAS) to identify single-stranded RNA (ssRNA) sequences bound by KH domains. We find that the FMRP KH0, KH1, and KH2 domains bind weakly to the ssRNA sequences suggesting that they may have evolved to bind more complex RNA structures. Additionally, we find that the RGG motif of human FMRP binds with a high affinity to an RNA G-quadruplex (GQ) structure that lacks single-stranded loops, double-stranded stems, or junctions. FMRP has been proposed to inhibit translation of target mRNAs in neurons. We investigated how human FMRP (hFMRP) regulates the translation of different mRNAs in rabbit reticulocyte lysate. We find that hFMRP inhibits different mRNAs to similar degrees. Furthermore, we observe inhibition is independent of the 5' cap-dependent initiation using . Finally, we dissected the RNA-binding domains of hFMRP and measured their inhibition of translation. We determined the RGG domain and C-terminal domain (CTD) are sufficient to inhibit translation while the KH domains do not inhibit mRNA translation. Interestingly, we see a correlation between ribosome binding and translation inhibition, suggesting the RGG-CTD tail of hFMRP may anchor FMRP to the ribosome during translation inhibition