The quadruplex r(CGG)n destabilizing cationic porphyrin TMPyP4 cooperates with hnRNPs to increase the translation efficiency of fragile X premutation mRNA

Abstract

The 5′ untranslated region of the FMR1 gene which normally includes 4–55 d(CGG) repeats expands to > 55–200 repeats in carriers of fragile X syndrome premutation. Although the levels of premutation FMR1 mRNA in carrier cells are 5–10-fold higher than normal, the amount of the product FMR protein is unchanged or reduced. We demonstrated previously that premutation r(CGG)n tracts formed quadruplex structures that impeded translation and lowered the efficiency of protein synthesis. Normal translation could be restored in vivo by the quadruplex r(CGG)n destabilizing action of CBF-A and hnRNP A2 proteins. Here we report that the quadruplex-interacting cationic porphyrin TMPyP4 by itself and in cooperation with CBF-A or hnRNP A2 also unfolded quadruplex r(CGG)n and increased the efficiency of translation of 5′-(CGG)99 containing reporter firefly (FL) mRNA. TMPyP4 destabilized in vitro a (CGG)33 intramolecular quadruplex structure and enhanced the translation of 5′-(CGG)99-FL mRNA in a rabbit reticulocyte lysate and in HEK293 cells. The efficiency of translation of (CGG)99-FL mRNA was additively increased in cells exposed to TMPyP4 together with CBF-A. Whereas low doses of TMPyP4, CBF-A or hnRNP A2 by themselves did not affect the in vivo utilization of (CGG)99-FL mRNA, introduction of TMPyP4 together with either protein synergistically augmented its translation efficiency.

Structural Diversity of Triplet Repeat RNAs

“…In a more recent NMR study, G/G mismatches were shown to exist in a dynamic equilibrium between the anti-and syn-conformations required to form two symmetric hydrogen bonds between the N1 and carbonyl oxygen atoms of two guanines (31). The CGG repeats were also reported to form G-quadruplexes in both RNA (11)(12)(13) and DNA (14,15). In the current study, we analyzed the structural features of CGG and other types of CNG repeats under identical conditions using biochemical and biophysical methods.…”

TMPyP4 Porphyrin Distorts RNA G-quadruplex Structures of the Disease-associated r(GGGGCC)n Repeat of the C9orf72 Gene and Blocks Interaction of RNA-binding Proteins

“…Similarly, TMPyP4 destabilized the G-quadruplex of both the DNA and the RNA (CGG)n repeats of FMR1, associated with premutation expansions of fragile X syndrome, fragile X-associated tremor ataxia, and fragile X premature ovarian insufficiency (34 -36). Disruption of both RNA G-quadruplexes (MT3-MMP and FMR1) by TMPyP4 led to enhanced levels of translation in model systems (33,35). Here we report the binding of TMPyP4 to the ALS-FTD r(GGGGCC)8 repeat using gel mobility shift assays, CD spectroscopy, and UV spectroscopy.…”

Mouse Models of Fragile X-Associated Tremor Ataxia

“…One possibility to explain this paradox is that the CGG expansion in the 5'-UTR of the transcript hampers the initiation of translation at the ribosome, possibly through the formation of quadruplex CGG secondary structures. 26 The cellular mechanisms that result in enhanced transcription of Fmr1 are unknown at present. Changes in the levels of Fmrp in the brain also seem to be brain region-specific with significant reductions in many regions of the brain, although expression remains relatively high in the hippocampus.…”