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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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Effects of (TTC)n repeats on RNA expression in yeast S. cerevisiae. (A) The scheme of linearized yeast reporter plasmid showing a yeast transcription cassette (shown in light grey) consisting of GAL1 promoter, 210-bp 5′ part, (TTC)n repeat, and 572-bp 3′ part. 5′ and 3′ probes for northern hybridization are shown. (B) Northern hybridization analysis of the repeat-containing and control RNA transcripts.
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Figure 8: Effects of (TTC)n repeats on RNA expression in yeast S. cerevisiae. (A) The scheme of linearized yeast reporter plasmid showing a yeast transcription cassette (shown in light grey) consisting of GAL1 promoter, 210-bp 5′ part, (TTC)n repeat, and 572-bp 3′ part. 5′ and 3′ probes for northern hybridization are shown. (B) Northern hybridization analysis of the repeat-containing and control RNA transcripts.

Mentions: Is this observation relevant for eukaryotic systems? To address this question, we analyzed the effect of (TTC)n repeats on RNA degradation in yeast cells. For this purpose, (GAA)n·(TTC)n repeats of different lengths or a control 188-bp non-repetitive sequence were cloned downstream of the GAL1 promoter such that (TTC)n runs appeared in the sense strand for transcription (Figure 8A). Upon transcription induction by the galactose, total RNA was isolated and analyzed by northern hybridization. Hybridization with the probe, corresponding to the 5′ part of the transcript, revealed the accumulation of short transcription products evidently truncated at the position of the (UUC)n repeat (Figure 8B). The accumulation of these truncations was clearly dependent on the repeat's length; they were non-existent at 20 repeats, evident at 35 repeats and became the main RNA species at 60 repeats. Hybridization with the 3′ probe revealed traces of RNAs, whose lengths corresponded to the distance from the (UUC)n run to the transcription terminator. We therefore believe that RNA truncations are likely caused by the UUC-mediated RNA degradation in yeast, similar to what was observed in bacteria. Since there is no known homolog of RNase E in yeast, it would be of prime interest to identify yeast RNase(s) responsible for this degradation. While UUC-mediated RNA degradation has not yet been observed in cultured mammalian cells (10), our data warrant revisiting this issue. It would also be interesting to study whether other pyrimidine-rich expandable repeats cause RNA degradation in vivo.Figure 8.


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)

Effects of (TTC)n repeats on RNA expression in yeast S. cerevisiae. (A) The scheme of linearized yeast reporter plasmid showing a yeast transcription cassette (shown in light grey) consisting of GAL1 promoter, 210-bp 5′ part, (TTC)n repeat, and 572-bp 3′ part. 5′ and 3′ probes for northern hybridization are shown. (B) Northern hybridization analysis of the repeat-containing and control RNA transcripts.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 8: Effects of (TTC)n repeats on RNA expression in yeast S. cerevisiae. (A) The scheme of linearized yeast reporter plasmid showing a yeast transcription cassette (shown in light grey) consisting of GAL1 promoter, 210-bp 5′ part, (TTC)n repeat, and 572-bp 3′ part. 5′ and 3′ probes for northern hybridization are shown. (B) Northern hybridization analysis of the repeat-containing and control RNA transcripts.
Mentions: Is this observation relevant for eukaryotic systems? To address this question, we analyzed the effect of (TTC)n repeats on RNA degradation in yeast cells. For this purpose, (GAA)n·(TTC)n repeats of different lengths or a control 188-bp non-repetitive sequence were cloned downstream of the GAL1 promoter such that (TTC)n runs appeared in the sense strand for transcription (Figure 8A). Upon transcription induction by the galactose, total RNA was isolated and analyzed by northern hybridization. Hybridization with the probe, corresponding to the 5′ part of the transcript, revealed the accumulation of short transcription products evidently truncated at the position of the (UUC)n repeat (Figure 8B). The accumulation of these truncations was clearly dependent on the repeat's length; they were non-existent at 20 repeats, evident at 35 repeats and became the main RNA species at 60 repeats. Hybridization with the 3′ probe revealed traces of RNAs, whose lengths corresponded to the distance from the (UUC)n run to the transcription terminator. We therefore believe that RNA truncations are likely caused by the UUC-mediated RNA degradation in yeast, similar to what was observed in bacteria. Since there is no known homolog of RNase E in yeast, it would be of prime interest to identify yeast RNase(s) responsible for this degradation. While UUC-mediated RNA degradation has not yet been observed in cultured mammalian cells (10), our data warrant revisiting this issue. It would also be interesting to study whether other pyrimidine-rich expandable repeats cause RNA degradation in vivo.Figure 8.

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