Date Published: May 21, 2019
Publisher: Public Library of Science
Author(s): Brett DeMarco, Snezana Stefanovic, Allison Williams, Kathryn R. Moss, Bart R. Anderson, Gary J. Bassell, Mihaela Rita Mihailescu, Yoon Ki Kim.
Fragile X syndrome, the most common inherited form of intellectual disability, is caused by the CGG trinucleotide expansion in the 5’-untranslated region of the Fmr1 gene on the X chromosome, which silences the expression of the fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA, which encodes for the postsynaptic density protein 95, and together with microRNA-125a to mediate the reversible inhibition of the PSD-95 mRNA translation in neurons. The miR-125a binding site within the PSD-95 mRNA 3’-untranslated region (UTR) is embedded in a G-rich region bound by FMRP, which we have previously demonstrated folds into two parallel G-quadruplex structures. The FMRP regulation of PSD-95 mRNA translation is complex, being mediated by its phosphorylation. While the requirement for FMRP in the regulation of PSD-95 mRNA translation is clearly established, the exact mechanism by which this is achieved is not known. In this study, we have shown that both unphosphorylated FMRP and its phosphomimic FMRP S500D bind to the PSD-95 mRNA G-quadruplexes with high affinity, whereas only FMRP S500D binds to miR-125a. These results point towards a mechanism by which, depending on its phosphorylation status, FMRP acts as a switch that potentially controls the stability of the complex formed by the miR-125a-guided RNA induced silencing complex (RISC) and PSD-95 mRNA.
Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, being caused by the silencing of the fragile X mental retardation (Fmr1) gene, which encodes for the fragile X mental retardation protein (FMRP). FMRP is a messenger RNA (mRNA) binding protein whose role in translation control is essential for normal brain function . FMRP has been proposed to function as a switch that regulates the local translation of neuronal mRNA targets in response to the cellular needs [2–4]. FMRP is part of a small family of RNA binding proteins, being most abundantly expressed in the brain and testes and characteristic in both fetal and adult tissues . FMRP shares about 60% of amino acid identity with its autosomal paralogs, FXR1P and FXR2P [6, 7]. Even though it was proposed that these paralogs could partially compensate for the loss of FMRP, it was later shown that the expression of both FXR1P and FXR2P is not altered in patients with fragile X syndrome or in Fmr1 knockout mice .
In this study we have shown that both FMRP ISO1 and its phosphomimic, FMRP S500D bind to the PSD-95 Q1 and Q2 G-quadruplexes with high affinity and specificity in vitro, with the phosphomimic targeting both structures with greater affinity than the unphosphorylated protein. The dissociation constants of FMRP S500D, Kd PSD-95 Q11234 = 66 ± 10 nM, Kd PSD-95 Q2 = 100 ± 17 nM), and FMRP ISO1 (Kd PSD-95 Q11234 = 128 ± 27 nM, Kd PSD-95 Q2 = 195 ± 16 nM, are the first reported binding values of FMRP to PSD-95 mRNA. We have previously reported that FMRP binds with high affinity to G-quadruplex structures formed within Shank1 mRNA , NR2B mRNA  and its own encoding Fmr1 mRNA, additional targets of the protein  Interestingly, for NR2B mRNA and one of the Shank1 mRNA G-quadruplexes a similar trend was observed where the affinity of FMRP S500D was higher, whereas there was no difference in affinity for the binding of FMRP ISO1 and FMRP S500D for its own mRNA. This difference might be related to the function of FMRP with respect to the particular mRNA, as FMRP is involved in the translation regulation of PSD-95, Shank1 and NR2B mRNAs, whereas in the case of Fmr1 mRNA, FMRP is involved in its alternative splicing to affect the production of the isoforms ISO1, ISO2 and ISO3 . Additionally, we have shown that the isolated PSD-95 Q1-Q2 G-quadruplexes are sufficient for the recognition of various isoforms of FMRP found within mouse brain lysates, including the phosphorylated FMRP (P-FMRP) (Fig 4), in agreement with our in vitro findings that both the unphosphorylated and phosphorylated FMRP target the G-quadruplex structures. Moreover, the structures adopted by these G-quadruplexes allow miR-125a to recognize its binding site, both in vitro and in mouse brain lysates (Fig 7).