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

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.