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.