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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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Effect of expanded (GAA)n repeats on RNA synthesis in E. coli. (A) Structure of the p185Δ plasmid, carrying (GAA)n·(TTC)n repeats in different orientations. (B) Northern blot analysis of GAA-containing mRNAs from the wild-type E. coli cells. Total RNA was hybridized with a probe, corresponding to either the 5′ part of the cat gene (upper panel) or RNAI, corresponding to the p15A origin (lower panel). (C) Quantitative analysis of the above data. (D) Normalized amounts of GAA-containing mRNAs from cells treated with chloramphenicol. (E) Normalized amount of the GAA-containing mRNAs from the rne(ts) mutant strain upon its cultivation at the non-permissive temperature.
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Figure 1: Effect of expanded (GAA)n repeats on RNA synthesis in E. coli. (A) Structure of the p185Δ plasmid, carrying (GAA)n·(TTC)n repeats in different orientations. (B) Northern blot analysis of GAA-containing mRNAs from the wild-type E. coli cells. Total RNA was hybridized with a probe, corresponding to either the 5′ part of the cat gene (upper panel) or RNAI, corresponding to the p15A origin (lower panel). (C) Quantitative analysis of the above data. (D) Normalized amounts of GAA-containing mRNAs from cells treated with chloramphenicol. (E) Normalized amount of the GAA-containing mRNAs from the rne(ts) mutant strain upon its cultivation at the non-permissive temperature.

Mentions: To study transcription through (GAA)n repeats of increasing lengths, they were cloned into a pACYC184-derived plasmid p185Δ downstream from an artificial trc promoter inside the cat gene (Figure 1A). This promoter is a hybrid between the 5′ part of the trp promoter and the 3′ part of the lacUV5 promoter, i.e. it is inducible by IPTG but is not a subject for catabolic repression (24). A strong terminator from the E. coli rrnB gene contributes to the stability of the cat mRNA. For the purpose of this study, we have deleted a 3′ portion of the cat gene, making the transcript approximately 500 nt shorter. To clone the longest repeats, it was crucial to achieve complete repression of the trc promoter, as the stability of the repeats appeared to decrease in transcribed areas (25). For this purpose, repeat-containing plasmids were co-transformed with the compatible pTrc99A plasmid (24) carrying lacIq repressor gene.Figure 1.


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)

Effect of expanded (GAA)n repeats on RNA synthesis in E. coli. (A) Structure of the p185Δ plasmid, carrying (GAA)n·(TTC)n repeats in different orientations. (B) Northern blot analysis of GAA-containing mRNAs from the wild-type E. coli cells. Total RNA was hybridized with a probe, corresponding to either the 5′ part of the cat gene (upper panel) or RNAI, corresponding to the p15A origin (lower panel). (C) Quantitative analysis of the above data. (D) Normalized amounts of GAA-containing mRNAs from cells treated with chloramphenicol. (E) Normalized amount of the GAA-containing mRNAs from the rne(ts) mutant strain upon its cultivation at the non-permissive temperature.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 1: Effect of expanded (GAA)n repeats on RNA synthesis in E. coli. (A) Structure of the p185Δ plasmid, carrying (GAA)n·(TTC)n repeats in different orientations. (B) Northern blot analysis of GAA-containing mRNAs from the wild-type E. coli cells. Total RNA was hybridized with a probe, corresponding to either the 5′ part of the cat gene (upper panel) or RNAI, corresponding to the p15A origin (lower panel). (C) Quantitative analysis of the above data. (D) Normalized amounts of GAA-containing mRNAs from cells treated with chloramphenicol. (E) Normalized amount of the GAA-containing mRNAs from the rne(ts) mutant strain upon its cultivation at the non-permissive temperature.
Mentions: To study transcription through (GAA)n repeats of increasing lengths, they were cloned into a pACYC184-derived plasmid p185Δ downstream from an artificial trc promoter inside the cat gene (Figure 1A). This promoter is a hybrid between the 5′ part of the trp promoter and the 3′ part of the lacUV5 promoter, i.e. it is inducible by IPTG but is not a subject for catabolic repression (24). A strong terminator from the E. coli rrnB gene contributes to the stability of the cat mRNA. For the purpose of this study, we have deleted a 3′ portion of the cat gene, making the transcript approximately 500 nt shorter. To clone the longest repeats, it was crucial to achieve complete repression of the trc promoter, as the stability of the repeats appeared to decrease in transcribed areas (25). For this purpose, repeat-containing plasmids were co-transformed with the compatible pTrc99A plasmid (24) carrying lacIq repressor gene.Figure 1.

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