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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 truncations caused by (TTC)n repeats are not due to the H-DNA formation. RNA from a plasmid with the (TTC)15 repeat and three mutant derivatives of this repeat, M1, M2 and M12, was analyzed by northern blot hybridization with the 5′ probe shown in Figure 1A.
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Figure 6: RNA truncations caused by (TTC)n repeats are not due to the H-DNA formation. RNA from a plasmid with the (TTC)15 repeat and three mutant derivatives of this repeat, M1, M2 and M12, was analyzed by northern blot hybridization with the 5′ probe shown in Figure 1A.

Mentions: Since (GAA)n·(TTC)n repeats are capable of forming triplex DNA structures, we first analyzed whether transcript truncations in the (TTC)n orientation were due to the formation of intramolecular triplexes (H-DNA). To check this hypothesis, we used an approach based on the fact that intramolecular triplexes require the mirror symmetry from a homopurine–homopyrimidine repeat. We generated mutants M1 and M2 that carried two C-to-T transitions in either the 5′ or 3′ half of the (TTC)15 repeat, respectively, thus destroying its mirror symmetry without changing the CT content. We have also generated a mutant M12, which combined substitutions from the M1 and M2 mutants together, restoring the mirror symmetry of the repeat. Northern blot analysis of transcripts from the resultant plasmids revealed that RNA truncations at TC-rich stretches remained the same for all three mutants and was indistinguishable from that in the non-interrupted (TTC)15 stretch (Figure 6). We therefore concluded that RNA truncations did not depend on the repeat's ability to form triplex structures. Since the RNA pattern did not change even in the M12 mutant where the (TTC)n repeat was significantly disturbed, we concluded that the perfect repetitive nature of the sequence is not required for transcript truncations.Figure 6.


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 truncations caused by (TTC)n repeats are not due to the H-DNA formation. RNA from a plasmid with the (TTC)15 repeat and three mutant derivatives of this repeat, M1, M2 and M12, was analyzed by northern blot hybridization with the 5′ probe shown in Figure 1A.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 6: RNA truncations caused by (TTC)n repeats are not due to the H-DNA formation. RNA from a plasmid with the (TTC)15 repeat and three mutant derivatives of this repeat, M1, M2 and M12, was analyzed by northern blot hybridization with the 5′ probe shown in Figure 1A.
Mentions: Since (GAA)n·(TTC)n repeats are capable of forming triplex DNA structures, we first analyzed whether transcript truncations in the (TTC)n orientation were due to the formation of intramolecular triplexes (H-DNA). To check this hypothesis, we used an approach based on the fact that intramolecular triplexes require the mirror symmetry from a homopurine–homopyrimidine repeat. We generated mutants M1 and M2 that carried two C-to-T transitions in either the 5′ or 3′ half of the (TTC)15 repeat, respectively, thus destroying its mirror symmetry without changing the CT content. We have also generated a mutant M12, which combined substitutions from the M1 and M2 mutants together, restoring the mirror symmetry of the repeat. Northern blot analysis of transcripts from the resultant plasmids revealed that RNA truncations at TC-rich stretches remained the same for all three mutants and was indistinguishable from that in the non-interrupted (TTC)15 stretch (Figure 6). We therefore concluded that RNA truncations did not depend on the repeat's ability to form triplex structures. Since the RNA pattern did not change even in the M12 mutant where the (TTC)n repeat was significantly disturbed, we concluded that the perfect repetitive nature of the sequence is not required for transcript truncations.Figure 6.

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