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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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RNase E is responsible for the cleavage of (UUC)n-containing RNAs. Northern blot analysis of repeat-containing RNA isolated from the JM109 (control) and N3431 (rnets) cells carrying the p185ΔCTT20 plasmid. Prior to RNA isolation, cells were incubated at permissive (30°C), semi-permissive (37°C) or non-permissive (43°C) temperatures for 1 h.
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Figure 7: RNase E is responsible for the cleavage of (UUC)n-containing RNAs. Northern blot analysis of repeat-containing RNA isolated from the JM109 (control) and N3431 (rnets) cells carrying the p185ΔCTT20 plasmid. Prior to RNA isolation, cells were incubated at permissive (30°C), semi-permissive (37°C) or non-permissive (43°C) temperatures for 1 h.

Mentions: RNase E, to the contrary, appeared to be crucial for the repeat-caused RNA cleavage. Since RNase E is an essential protein, we used a temperature-sensitive rne-3071(ts) mutant and followed the fate of (UUC)n-containing RNAs upon shifting the logarithmic cells to different temperatures. Figure 7 shows a clear-cut decrease in the amount of truncated RNA fragments at the non-permissive compared to permissive temperatures. RNase E is the key component of the so-called degradosome, which is involved in the endonucleolytic degradation of bacterial mRNA. We believe, therefore, that RNA cleavage within (UUC)n repeats by the bacterial degradosome is responsible for the accumulation of truncated RNAs, which could be mistaken for the products of premature transcription attenuation.Figure 7.


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)

RNase E is responsible for the cleavage of (UUC)n-containing RNAs. Northern blot analysis of repeat-containing RNA isolated from the JM109 (control) and N3431 (rnets) cells carrying the p185ΔCTT20 plasmid. Prior to RNA isolation, cells were incubated at permissive (30°C), semi-permissive (37°C) or non-permissive (43°C) temperatures for 1 h.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 7: RNase E is responsible for the cleavage of (UUC)n-containing RNAs. Northern blot analysis of repeat-containing RNA isolated from the JM109 (control) and N3431 (rnets) cells carrying the p185ΔCTT20 plasmid. Prior to RNA isolation, cells were incubated at permissive (30°C), semi-permissive (37°C) or non-permissive (43°C) temperatures for 1 h.
Mentions: RNase E, to the contrary, appeared to be crucial for the repeat-caused RNA cleavage. Since RNase E is an essential protein, we used a temperature-sensitive rne-3071(ts) mutant and followed the fate of (UUC)n-containing RNAs upon shifting the logarithmic cells to different temperatures. Figure 7 shows a clear-cut decrease in the amount of truncated RNA fragments at the non-permissive compared to permissive temperatures. RNase E is the key component of the so-called degradosome, which is involved in the endonucleolytic degradation of bacterial mRNA. We believe, therefore, that RNA cleavage within (UUC)n repeats by the bacterial degradosome is responsible for the accumulation of truncated RNAs, which could be mistaken for the products of premature transcription attenuation.Figure 7.

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