Nucleic Acids Research Advance Access originally published online on August 23, 2007
Nucleic Acids Research 2007 35(17):5775-5788; doi:10.1093/nar/gkm636
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Nucleic Acids Research, 2007, Vol. 35, No. 17 5775-5788
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
The tetraplex (CGG)n destabilizing proteins hnRNP A2 and CBF-A enhance the in vivo translation of fragile X premutation mRNA
1Department of Biochemistry, Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa 31096, Israel and 2Department of Biochemistry and Molecular Medicine, School of Medicine, University of California Davis, One Shields Avenue, Davis, CA 95616-8635, USA
*To whom correspondence should be addressed. Tel: 972 4 829 5328; Fax: 972 4 851 0735; Email: mickey{at}tx.technion.ac.il
Received April 8, 2007. Revised July 31, 2007. Accepted August 1, 2007.
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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