The tetraplex (CGG)n destabilizing proteins hnRNP A2 and CBF-A enhance the in vivo translation of fragile X premutation mRNA

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Abstract
Expansion of a (CGG) n sequence in the 5′-UTR of the FMR1 gene to > 200–2000 repeats abolishes its transcription and initiates fragile X syndrome (FXS). By contrast, levels of FMR1 mRNA are 5–10-fold higher in FXS premutation carriers of > 55–200 repeats than in normal subjects. Lack of a corresponding increase in the amount of the product FMRP protein in carrier cells suggest that (CGG) > 55–200 tracts thwart translation. Here we report that a (CGG) 99 sequence positioned upstream to reporter firefly (FL) gene selectively diminished mRNA translation in coupled and separate T7 promoter-driven in vitro transcription and translation systems. The (CGG) 99 tract similarly depressed mRNA utilization in HEK293 human cells transfected with plasmids bearing FMR1 promoter-driven FL gene. A (CGG) 33 RNA tract formed a largely RNase T1-resistant intramolecular secondary structure in the presence of K + ions. Expression of the quadruplex (CGG) n disrupting proteins hnRNP A2 or CBF-A in HEK293 cells significantly elevated the efficacy of (CGG) 99 FL mRNA translation whereas hnRNP A2 or CBF-A mutants lacking quadruplex (CGG) n disrupting activity did not. Taken together, our results suggest that secondary structures of (CGG) n in mRNA obstruct its translation and that quadruplex-disrupting proteins alleviate the translational block.
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