CGG Repeats in the 5’UTR of FMR1 RNA Regulate Translation of Other RNAs Localized in the Same RNA Granules

Abstract

CGG repeats in the 5’UTR of Fragile X Mental Retardation 1 (FMR1) RNA mediate RNA localization and translation in granules. Large expansions of CGG repeats (> 200 repeats) in FMR1, referred to as full mutations, are associated with fragile X syndrome (FXS). Smaller expansions (55–200 repeats), referred to as premutations, are associated with fragile X tremor ataxia syndrome (FXTAS) and fragile X premature ovarian insufficiency (FXPOI). TMPyP4 is a porphyrin ring compound that destabilizes CGG repeat RNA secondary structure. Here we show that exogenous CGG repeat RNA by itself, lacking the FMRP ORF, microinjected into hippocampal neurons is localized in RNA granules and inhibits translation of ARC RNA, which is localized in the same granules. TMPyP4 rescues translation of ARC RNA in granules. We also show that in human premutation fibroblasts with endogenous CGG repeat expansions in the FMR1 gene, translation of ARC RNA is inhibited and calcium homeostasis is disrupted and both phenotypes are rescued by TMPyP4. Inhibition of granule translation by expanded CGG repeats and rescue of granule translation by TMPy4, represent potential pathogenic mechanism and therapeutic strategy, respectively, for FXTAS and FXPOI.

Multifarious Functions of the Fragile X Mental Retardation Protein

“…1), although FMRP has long been associated with mRNA throughout its lifecycle, including splicing, editing, trafficking and stability [6,11,12,29,30]. FMRP selectively associates with a subset of mRNAs: the candidate target list is long, but altered protein levels have been established for only a tiny handful of proteins (Table 2) [29,31]. Indeed, proteomic screens suggest that the number of protein changes in both mouse and Drosophila FXS models is surprisingly small, and many of these changes may be indirect, since they don’t align well with mRNA-binding data [32].…”

“…In Drosophila , FMRP-L5 binding prevents tRNA and elongation factor ribosome association to suppress translation. A recent study in human FXS patient cells suggests translation repression also arises from FMR1 5'UTR CGG binding to activity-regulated cytoskeleton associated protein (ARC) transcripts in mobile ribosome granules, inhibiting mRNA localization/translation (Table 2) [29,37,38]. In mice, telomere repeat-binding factor 2 (TRF2-S) binding to target mRNAs facilitates axonal delivery, which is antagonized by FMRP binding the TRF2-S GAR domain [39].…”

“…FMRP indirectly controls presynaptic N-type Ca 2+ channel levels (Ca v 2.2) via proteasomal degradation in human cells [18]. Downstream of channels, FMRP also regulates calcium binding protein (CaBP) function, through both RNA- and protein-binding mechanisms [10,29,56,57]. Moreover, the Drosophila FXS model shows reduced Calmodulin and Calbindin CaBP mRNA levels, resulting in the impaired sequestration of cytosolic Ca 2+ (Table 2) [58].…”

“…FMRP also modulates calcium homeostasis in non-canonical mechanisms. As referenced above, in human cells the FMR1 5'UTR CGG expansion interacts with ARC mRNA in mobile ribosomal granules, which also disrupts Ca 2+ homeostasis by misregulating CaBP mRNAs that localize to these granules in a similar translation misregulation mechanism [29]. Using a drug that destabilizes the FMR1 CGG repeats (TMPyP4), normal Ca 2+ homeostasis can be restored, indicating some relationship between the FXS trinucleotide expansion and neural Ca 2+ dynamics [29].…”

“…As referenced above, in human cells the FMR1 5'UTR CGG expansion interacts with ARC mRNA in mobile ribosomal granules, which also disrupts Ca 2+ homeostasis by misregulating CaBP mRNAs that localize to these granules in a similar translation misregulation mechanism [29]. Using a drug that destabilizes the FMR1 CGG repeats (TMPyP4), normal Ca 2+ homeostasis can be restored, indicating some relationship between the FXS trinucleotide expansion and neural Ca 2+ dynamics [29]. Added to this complexity, FMRP-dependent Ca 2+ signaling is altered differentially in excitatory versus inhibitory neurons as they mature during the early-use critical period in the Drosophila FXS model [1].…”