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Effects of Friedreich's ataxia (GAA)n*(TTC)n repeats on RNA synthesis and stability.

Krasilnikova MM, Kireeva ML, Petrovic V, Knijnikova N, Kashlev M, Mirkin SM - Nucleic Acids Res. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.

ABSTRACT
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.

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RNA polymerase progression is blocked by the (GAA)n repeats in vitro. (A) The immobilized elongation complexes were ligated to the linear fragment containing the repeat (GAA or TTC in the sense strand) or the control sequence. The 45-nt RNA in the starting EC and the 300-nt run-off (RO) product are indicated by arrows. (B) Quantitative analyses of data shown in (A); the fraction of the starting elongation complexes that reached the end of the template reflects the transcription efficiency. (C) The ECs obtained as in (A) were chased for 5 min with 1 mM NTPs, the KCl concentration was adjusted to 1 M, and the complexes were incubated in high salt for an additional 5 min. Then, the supernatant was removed (S, odd lanes) and loaded side-by-side with the unfractionated reaction mix (T, even lanes). The run-off products are indicated by arrows; the range of the transcripts from the TECs blocked by the repeat is shown by a red line.
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Figure 4: RNA polymerase progression is blocked by the (GAA)n repeats in vitro. (A) The immobilized elongation complexes were ligated to the linear fragment containing the repeat (GAA or TTC in the sense strand) or the control sequence. The 45-nt RNA in the starting EC and the 300-nt run-off (RO) product are indicated by arrows. (B) Quantitative analyses of data shown in (A); the fraction of the starting elongation complexes that reached the end of the template reflects the transcription efficiency. (C) The ECs obtained as in (A) were chased for 5 min with 1 mM NTPs, the KCl concentration was adjusted to 1 M, and the complexes were incubated in high salt for an additional 5 min. Then, the supernatant was removed (S, odd lanes) and loaded side-by-side with the unfractionated reaction mix (T, even lanes). The run-off products are indicated by arrows; the range of the transcripts from the TECs blocked by the repeat is shown by a red line.

Mentions: Figure 4A shows that the (GAA)57 repeat in the sense strand severely inhibited transcription under these experimental conditions. While roughly 40% of starting EC45s formed run-off products upon chase with all four NTPs on the control and (TTC)n-encoding templates (Figure 4A (lanes 2 and 6) and B), only 7% of those complexes were able to complete transcription through the repetitive stretch on (GAA)n-encoding templates (Figure 4A (lane 4) and B). Overexposure of the gel revealed an array of shorter RNAs spanning the entire repetitive run (Figure 4A, lane 7). Therefore, the repeats caused either transcription arrest or termination.Figure 4.


Effects of Friedreich's ataxia (GAA)n*(TTC)n repeats on RNA synthesis and stability.

Krasilnikova MM, Kireeva ML, Petrovic V, Knijnikova N, Kashlev M, Mirkin SM - Nucleic Acids Res. (2007)

RNA polymerase progression is blocked by the (GAA)n repeats in vitro. (A) The immobilized elongation complexes were ligated to the linear fragment containing the repeat (GAA or TTC in the sense strand) or the control sequence. The 45-nt RNA in the starting EC and the 300-nt run-off (RO) product are indicated by arrows. (B) Quantitative analyses of data shown in (A); the fraction of the starting elongation complexes that reached the end of the template reflects the transcription efficiency. (C) The ECs obtained as in (A) were chased for 5 min with 1 mM NTPs, the KCl concentration was adjusted to 1 M, and the complexes were incubated in high salt for an additional 5 min. Then, the supernatant was removed (S, odd lanes) and loaded side-by-side with the unfractionated reaction mix (T, even lanes). The run-off products are indicated by arrows; the range of the transcripts from the TECs blocked by the repeat is shown by a red line.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851639&req=5

Figure 4: RNA polymerase progression is blocked by the (GAA)n repeats in vitro. (A) The immobilized elongation complexes were ligated to the linear fragment containing the repeat (GAA or TTC in the sense strand) or the control sequence. The 45-nt RNA in the starting EC and the 300-nt run-off (RO) product are indicated by arrows. (B) Quantitative analyses of data shown in (A); the fraction of the starting elongation complexes that reached the end of the template reflects the transcription efficiency. (C) The ECs obtained as in (A) were chased for 5 min with 1 mM NTPs, the KCl concentration was adjusted to 1 M, and the complexes were incubated in high salt for an additional 5 min. Then, the supernatant was removed (S, odd lanes) and loaded side-by-side with the unfractionated reaction mix (T, even lanes). The run-off products are indicated by arrows; the range of the transcripts from the TECs blocked by the repeat is shown by a red line.
Mentions: Figure 4A shows that the (GAA)57 repeat in the sense strand severely inhibited transcription under these experimental conditions. While roughly 40% of starting EC45s formed run-off products upon chase with all four NTPs on the control and (TTC)n-encoding templates (Figure 4A (lanes 2 and 6) and B), only 7% of those complexes were able to complete transcription through the repetitive stretch on (GAA)n-encoding templates (Figure 4A (lane 4) and B). Overexposure of the gel revealed an array of shorter RNAs spanning the entire repetitive run (Figure 4A, lane 7). Therefore, the repeats caused either transcription arrest or termination.Figure 4.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus