Nucleic Acids Research Advance Access published online on January 30, 2007
Nucleic Acids Research, doi:10.1093/nar/gkl1140
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Molecular Biology |
Effects of Friedreich's ataxia (GAA)n·(TTC)n repeats on RNA synthesis and stability
1Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA, 2NCI Center for Cancer Research, Frederick, MD 21702, USA and 3Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA
*To whom correspondence should be addressed. Tel: +1 617 627 4794; Fax: +1 617 627 3805; E-mail: sergei.mirkin{at}tufts.edu
Received October 25, 2006. Revised December 6, 2006. Accepted December 14, 2006.
Expansions of (GAA)n repeats within the first intron of the frataxin gene reduce its expression, resulting in a hereditary neurodegenerative disorder, Friedreich's ataxia. While it is generally believed that expanded (GAA)n repeats block transcription elongation, fine mechanisms responsible for gene repression are not fully understood. To follow the effects of (GAA)n·(TTC)n repeats on gene expression, we have chosen E. coli as a convenient model system. (GAA)n·(TTC)n repeats were cloned into bacterial plasmids in both orientations relative to a promoter, and their effects on transcription and RNA stability were evaluated both in vitro and in vivo. Expanded (GAA)n repeats in the sense strand for transcription caused a significant decrease in the mRNA levels in vitro and in vivo. This decrease was likely due to the tardiness of the RNA polymerase within expanded (GAA)n runs but was not accompanied by the enzyme's dissociation and premature transcription termination. Unexpectedly, positioning of normal- and carrier-size (TTC)n repeats into the sense strand for transcription led to the appearance of RNA transcripts that were truncated within those repetitive runs in vivo. We have determined that these RNA truncations are consistent with cleavage of the full-sized mRNAs at (UUC)n runs by the E. coli degradosome.
Present address: Sergei M. Mirkin, Department of Biology, Tufts University, Medford, MA 02155, USA.
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