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Knocking down gene function with an RNA aptamer expressed as part of an intron.

Wang S, Zhao X, Suran R, Vogt VM, Lis JT, Shi H - Nucleic Acids Res. (2010)

Bottom Line: We developed a powerful expression system to produce aptamers and other types of functional RNA in yeast to examine their effects.Utilizing the intron homing process, the aptamer-coding sequences were integrated into hundreds of rRNA genes, and the aptamers were transcribed at high levels by RNA polymerase I without any additional promoter being introduced into the cell.As HSF1 enables and promotes malignant growth and metastasis in mammals, and this aptamer binds yeast HSF1 and its mammalian ortholog with equal affinity, the results presented here attest to the potential of this aptamer as a specific and effective inhibitor of HSF1 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University at Albany, State University of New York, Albany, NY 12222, USA.

ABSTRACT
We developed a powerful expression system to produce aptamers and other types of functional RNA in yeast to examine their effects. Utilizing the intron homing process, the aptamer-coding sequences were integrated into hundreds of rRNA genes, and the aptamers were transcribed at high levels by RNA polymerase I without any additional promoter being introduced into the cell. We used this system to express an aptamer against the heat shock factor 1 (HSF1), a conserved transcription factor responsible for mobilizing specific genomic expression programs in response to stressful conditions such as elevated temperature. We observed a temperature sensitive growth retardation phenotype and specific decrease of heat shock gene expression. As HSF1 enables and promotes malignant growth and metastasis in mammals, and this aptamer binds yeast HSF1 and its mammalian ortholog with equal affinity, the results presented here attest to the potential of this aptamer as a specific and effective inhibitor of HSF1 activity.

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Molecular phenotype of the inhibitory HSF aptamer. (A) Effect of aptamer monomer (M) on the level of HS genes measured by conventional RT–PCR. ‘R1’ is a strain expressing the antisense sequence of the aptamer monomer. HS (20 min at 39°C). (B) Effect of both aptamer monomer (M) and dimer (D) on the level of HS genes measured by RT–qPCR. ‘R2’ is a strain expressing the antisense sequence of the aptamer dimer. The RNA level for each gene is presented as the ratio to the full HS induction level in the antisense control strain, which is set to 1. The expression level for each gene is normalized to that of ADH1. (We also used U6 to normalize the data, and the data sets were consistent with each other.) The error bars show the standard error from RT–qPCR experiments using three independently heat-shocked yeast RNA preparations from the same strain. In both panels the parental strain used was W303-1A.
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Figure 4: Molecular phenotype of the inhibitory HSF aptamer. (A) Effect of aptamer monomer (M) on the level of HS genes measured by conventional RT–PCR. ‘R1’ is a strain expressing the antisense sequence of the aptamer monomer. HS (20 min at 39°C). (B) Effect of both aptamer monomer (M) and dimer (D) on the level of HS genes measured by RT–qPCR. ‘R2’ is a strain expressing the antisense sequence of the aptamer dimer. The RNA level for each gene is presented as the ratio to the full HS induction level in the antisense control strain, which is set to 1. The expression level for each gene is normalized to that of ADH1. (We also used U6 to normalize the data, and the data sets were consistent with each other.) The error bars show the standard error from RT–qPCR experiments using three independently heat-shocked yeast RNA preparations from the same strain. In both panels the parental strain used was W303-1A.

Mentions: As shown in Figure 4A, using RT–PCR, we observed a significant decrease in heat-induced expression of all three typical HS genes in the strain expressing the AptHSF monomer. The difference in ratios of mRNA levels under HS and NHS conditions for the aptamer and antisense strains for each gene analyzed indicated a downregulation of HSF1 activity by the aptamer. In contrast, the expression level of HSP12 was not changed under either HS or NHS conditions. To confirm this result and compare the efficacy of the monomer and dimer, we used quantitative PCR (qPCR) to measure the mRNA level of the same three HS genes in both monomer- and dimer-expressing strains. Figure 4B shows a more pronounced decrease in the level of these mRNAs in the dimer-expressing strain than in the monomer-expressing strain under both HS and NHS conditions. Notably, the aptamer did not entirely inhibit HSF function, as in some cases an increase in transcription still occurred after HS. However, the resulting level of transcription apparently did not produce sufficient proteins to enable survival of the HS. In this experiment, we used the RNA polymerase II-driven ethanol-inducible gene ADH1 and the Pol III-driven constitutive gene U6 to normalize the data. These genes were not induced by HS and were not inhibited by the aptamer.Figure 4.


Knocking down gene function with an RNA aptamer expressed as part of an intron.

Wang S, Zhao X, Suran R, Vogt VM, Lis JT, Shi H - Nucleic Acids Res. (2010)

Molecular phenotype of the inhibitory HSF aptamer. (A) Effect of aptamer monomer (M) on the level of HS genes measured by conventional RT–PCR. ‘R1’ is a strain expressing the antisense sequence of the aptamer monomer. HS (20 min at 39°C). (B) Effect of both aptamer monomer (M) and dimer (D) on the level of HS genes measured by RT–qPCR. ‘R2’ is a strain expressing the antisense sequence of the aptamer dimer. The RNA level for each gene is presented as the ratio to the full HS induction level in the antisense control strain, which is set to 1. The expression level for each gene is normalized to that of ADH1. (We also used U6 to normalize the data, and the data sets were consistent with each other.) The error bars show the standard error from RT–qPCR experiments using three independently heat-shocked yeast RNA preparations from the same strain. In both panels the parental strain used was W303-1A.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926621&req=5

Figure 4: Molecular phenotype of the inhibitory HSF aptamer. (A) Effect of aptamer monomer (M) on the level of HS genes measured by conventional RT–PCR. ‘R1’ is a strain expressing the antisense sequence of the aptamer monomer. HS (20 min at 39°C). (B) Effect of both aptamer monomer (M) and dimer (D) on the level of HS genes measured by RT–qPCR. ‘R2’ is a strain expressing the antisense sequence of the aptamer dimer. The RNA level for each gene is presented as the ratio to the full HS induction level in the antisense control strain, which is set to 1. The expression level for each gene is normalized to that of ADH1. (We also used U6 to normalize the data, and the data sets were consistent with each other.) The error bars show the standard error from RT–qPCR experiments using three independently heat-shocked yeast RNA preparations from the same strain. In both panels the parental strain used was W303-1A.
Mentions: As shown in Figure 4A, using RT–PCR, we observed a significant decrease in heat-induced expression of all three typical HS genes in the strain expressing the AptHSF monomer. The difference in ratios of mRNA levels under HS and NHS conditions for the aptamer and antisense strains for each gene analyzed indicated a downregulation of HSF1 activity by the aptamer. In contrast, the expression level of HSP12 was not changed under either HS or NHS conditions. To confirm this result and compare the efficacy of the monomer and dimer, we used quantitative PCR (qPCR) to measure the mRNA level of the same three HS genes in both monomer- and dimer-expressing strains. Figure 4B shows a more pronounced decrease in the level of these mRNAs in the dimer-expressing strain than in the monomer-expressing strain under both HS and NHS conditions. Notably, the aptamer did not entirely inhibit HSF function, as in some cases an increase in transcription still occurred after HS. However, the resulting level of transcription apparently did not produce sufficient proteins to enable survival of the HS. In this experiment, we used the RNA polymerase II-driven ethanol-inducible gene ADH1 and the Pol III-driven constitutive gene U6 to normalize the data. These genes were not induced by HS and were not inhibited by the aptamer.Figure 4.

Bottom Line: We developed a powerful expression system to produce aptamers and other types of functional RNA in yeast to examine their effects.Utilizing the intron homing process, the aptamer-coding sequences were integrated into hundreds of rRNA genes, and the aptamers were transcribed at high levels by RNA polymerase I without any additional promoter being introduced into the cell.As HSF1 enables and promotes malignant growth and metastasis in mammals, and this aptamer binds yeast HSF1 and its mammalian ortholog with equal affinity, the results presented here attest to the potential of this aptamer as a specific and effective inhibitor of HSF1 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University at Albany, State University of New York, Albany, NY 12222, USA.

ABSTRACT
We developed a powerful expression system to produce aptamers and other types of functional RNA in yeast to examine their effects. Utilizing the intron homing process, the aptamer-coding sequences were integrated into hundreds of rRNA genes, and the aptamers were transcribed at high levels by RNA polymerase I without any additional promoter being introduced into the cell. We used this system to express an aptamer against the heat shock factor 1 (HSF1), a conserved transcription factor responsible for mobilizing specific genomic expression programs in response to stressful conditions such as elevated temperature. We observed a temperature sensitive growth retardation phenotype and specific decrease of heat shock gene expression. As HSF1 enables and promotes malignant growth and metastasis in mammals, and this aptamer binds yeast HSF1 and its mammalian ortholog with equal affinity, the results presented here attest to the potential of this aptamer as a specific and effective inhibitor of HSF1 activity.

Show MeSH
Related in: MedlinePlus