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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 normal-size (TTC)n repeats on RNA synthesis in E. coli cells. Northern blot analysis of UUC-containing mRNAs from the wild-type E. coli cells. Hybridization was carried out with either the 5′ or 3′ part (Figure 1A) of the repeat-containing transcript.
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Figure 5: Effect of normal-size (TTC)n repeats on RNA synthesis in E. coli cells. Northern blot analysis of UUC-containing mRNAs from the wild-type E. coli cells. Hybridization was carried out with either the 5′ or 3′ part (Figure 1A) of the repeat-containing transcript.

Mentions: Based on the above in vitro data, one should not expect transcription inhibition by (TTC)n repeats in the sense strand for transcription. Strikingly, however, we observed that transcription of plasmids carrying normal-size (TTC)n repeats in the sense strand resulted in the appearance of specifically truncated mRNAs (Figure 5). The lengths of the shortened RNA species indicated that truncations occurred around (UUC)n repeats. Furthermore, the fraction of truncated products rapidly increased with an increase in the repeat's length whereas the fraction of the full-size transcripts concurrently decreased. This length dependence strongly suggested that (TTC)n repeats in the sense strand were indeed responsible for the RNA truncation. Hybridization of the same blots with the probe corresponding to the 3′ end of the cat mRNA (Figure 5B) revealed only full-size RNA transcripts but not the 3′ truncations. This could either be due to the lack of transcription beyond short (TTC)n runs or an endonucleolytic cleavage of the repeat-containing transcripts followed by the rapid degradation of their 3′ halves.Figure 5.


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 normal-size (TTC)n repeats on RNA synthesis in E. coli cells. Northern blot analysis of UUC-containing mRNAs from the wild-type E. coli cells. Hybridization was carried out with either the 5′ or 3′ part (Figure 1A) of the repeat-containing transcript.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 5: Effect of normal-size (TTC)n repeats on RNA synthesis in E. coli cells. Northern blot analysis of UUC-containing mRNAs from the wild-type E. coli cells. Hybridization was carried out with either the 5′ or 3′ part (Figure 1A) of the repeat-containing transcript.
Mentions: Based on the above in vitro data, one should not expect transcription inhibition by (TTC)n repeats in the sense strand for transcription. Strikingly, however, we observed that transcription of plasmids carrying normal-size (TTC)n repeats in the sense strand resulted in the appearance of specifically truncated mRNAs (Figure 5). The lengths of the shortened RNA species indicated that truncations occurred around (UUC)n repeats. Furthermore, the fraction of truncated products rapidly increased with an increase in the repeat's length whereas the fraction of the full-size transcripts concurrently decreased. This length dependence strongly suggested that (TTC)n repeats in the sense strand were indeed responsible for the RNA truncation. Hybridization of the same blots with the probe corresponding to the 3′ end of the cat mRNA (Figure 5B) revealed only full-size RNA transcripts but not the 3′ truncations. This could either be due to the lack of transcription beyond short (TTC)n runs or an endonucleolytic cleavage of the repeat-containing transcripts followed by the rapid degradation of their 3′ halves.Figure 5.

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