Arginines of the RGG box regulate FMRP association with polyribosomes and mRNA

Abstract

Fragile X syndrome is caused by the loss of expression of the fragile X mental retardation protein, FMRP. FMRP is an RNA-binding protein that is highly expressed in neurons and undergoes multiple post-translational modifications including methylation on arginine. FMRP is methylated on the high-affinity RNA-binding motif, the RGG box, at positions 533, 538, 543 and 545 of murine FMRP. To identify the arginines important for FMRP function, we examined their role in polyribosome and mRNA association. We found that arginines 533 and 538 were required for normal FMRP polyribosome association whereas all four arginines played a role in RNA binding, depending on the identity of the RNA. The model G-quadruplex RNA sc1 required arginines 533 and 538 for normal association with FMRP, whereas AATYK mRNA did not. In vitro methylation of FMRP-bearing arginine substitutions inhibited sc1 binding but not AATYK binding. In addition, we found that PRMT1 co-immunoprecipitated with FMRP isolated from cells and that siRNAs directed against PRMT1 led to reduced FMRP methylation. Thus, two lines of experimentation demonstrate that PRMT1 acts on FMRP in cells. In summary, we provide evidence for the important role of the RGG box in polyribosome association. We also demonstrate for the first time that the different arginines of the RGG box are important for the binding of different RNAs. Finally, we show that PRMT1 methylates FMRP in cells, suggesting a model where methylation of the RGG box modulates either the quantity or the identity of the RNAs bound by FMRP.

Crystal structure reveals specific recognition of a G-quadruplex RNA by a β-turn in the RGG motif of FMRP

“…51). It is intriguing that Arg10 and Arg15, involved in polyribosome binding (14), are among four arginines targeted for methylation in FMRP and their methylation decreases RNA binding affinity (14,52).…”

“…Notably, binding of FMRP to G-rich RNAs in vitro requires only the RGG motif, which specifically interacts with natural and in vitro selected G-quadruplex-containing RNAs such as a 35-nucleotide sc1 RNA (21,(26)(27)(28)(29). Recent studies showed that G-quadruplexes facilitate mRNA interactions with ribosome-bound FMRP (30), whereas the RGG motif, in addition to mRNA binding, contributes to association with ribosomes and proteins and translational control (14,(31)(32)(33)(34). The RGG motif is well conserved in FMRP of vertebrates but differs significantly from…”

“…Loss of repression of these mRNAs is associated with alterations in synaptic plasticity and dendritic spine dynamics thought to underlie the manifestation of FXS (13). The mechanisms of the FMRPdependent translational repression have been suggested to include stalling of ribosomes by direct interactions of FMRP with mRNA (12) involving the RGG box (14) and KH2 domain (15). Individual contribution of each domain to RNA binding and translational repression and their functional relationship remain to be understood.…”

Translational Repression of the Disintegrin and Metalloprotease ADAM10 by a Stable G-quadruplex Secondary Structure in Its 5′-Untranslated Region

“…The RGG box of FMRP was shown to be responsible for the interaction with the G-quadruplex secondary structure in the MAP1B 5Ј-UTR (43). Therefore, methylation of RGG motifs in FMRP by PMRT1 might regulate the interaction of FMRP with polyribosomes and G-quadruplex-containing mRNAs (81). In addition, members of the hnRNP A family were reported to destabilize G-quadruplex structures by a so-far unknown mechanism (44,82).…”

RS and RGG repeats as primitive proteins at the transition between the RNA and RNP worlds

“…Proteins that contain simple unstructured repeats have been found to act as RNA chaperones 5,6 and the intrinsically unstructured regions of several RNA chaperones have been proposed to possess such chaperone activity. 4,5 RS domains consist of several (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) copies of the RS dipeptide, irregularly interrupted by other amino acids. RS domains can directly bind RNA and themselves 7,8 in a rather non-specific manner.…”