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High-throughput genetic and gene expression analysis of the RNAPII-CTD reveals unexpected connections to SRB10/CDK8.

Aristizabal MJ, Negri GL, Benschop JJ, Holstege FC, Krogan NJ, Kobor MS - PLoS Genet. (2013)

Bottom Line: Truncating the CTD altered RNAPII occupancy, leading to not only decreases, but also increases in mRNA levels.This suggested a positive role of Cdk8 in relationship to RNAPII, which contrasted with the observed negative role at the activated INO1 gene.Here, loss of CDK8 suppressed the reduced mRNA expression and RNAPII occupancy levels of CTD truncation mutants.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
The C-terminal domain (CTD) of RNA polymerase II (RNAPII) is composed of heptapeptide repeats, which play a key regulatory role in gene expression. Using genetic interaction, chromatin immunoprecipitation followed by microarrays (ChIP-on-chip) and mRNA expression analysis, we found that truncating the CTD resulted in distinct changes to cellular function. Truncating the CTD altered RNAPII occupancy, leading to not only decreases, but also increases in mRNA levels. The latter were largely mediated by promoter elements and in part were linked to the transcription factor Rpn4. The mediator subunit Cdk8 was enriched at promoters of these genes, and its removal not only restored normal mRNA and RNAPII occupancy levels, but also reduced the abnormally high cellular amounts of Rpn4. This suggested a positive role of Cdk8 in relationship to RNAPII, which contrasted with the observed negative role at the activated INO1 gene. Here, loss of CDK8 suppressed the reduced mRNA expression and RNAPII occupancy levels of CTD truncation mutants.

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INO1 expression and RNAPII association defects of rpb1-CTD11 mutants were suppressed by deleting CDK8.Cells were grown in inositol containing media (200 µM) to constitute the uninduced sample, and shifted to inositol deplete media for 4 hrs to constitute the induced sample. (A) qRT-PCR analysis of INO1 expression revealed a restoration of expression upon loss of CDK8. INO1 mRNA levels were normalized to ACT1 levels. (B) The sensitivity of CTD truncation mutants containing 11 or 12 repeats to growth in media lacking inositol was suppressed by deleting CDK8. (C) ChIP analysis of Rpb3 binding along the INO1 gene. Asterisks indicate induced conditions. Rpb3 enrichment along the INO1 gene was normalized to an intergenic region of chromosome V. Error bars represent standard deviations of values from three replicates.
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pgen-1003758-g007: INO1 expression and RNAPII association defects of rpb1-CTD11 mutants were suppressed by deleting CDK8.Cells were grown in inositol containing media (200 µM) to constitute the uninduced sample, and shifted to inositol deplete media for 4 hrs to constitute the induced sample. (A) qRT-PCR analysis of INO1 expression revealed a restoration of expression upon loss of CDK8. INO1 mRNA levels were normalized to ACT1 levels. (B) The sensitivity of CTD truncation mutants containing 11 or 12 repeats to growth in media lacking inositol was suppressed by deleting CDK8. (C) ChIP analysis of Rpb3 binding along the INO1 gene. Asterisks indicate induced conditions. Rpb3 enrichment along the INO1 gene was normalized to an intergenic region of chromosome V. Error bars represent standard deviations of values from three replicates.

Mentions: A previously characterized phenotype of CTD truncation mutants is reduced activation of INO1 and GAL10 upon switching to inducing conditions. Therefore, we investigated if loss of CDK8 could also suppress these expression defects of CTD truncation mutants [7]. Focusing on INO1, a gene important for the synthesis of inositol and survival in response to inositol starvation, we measured INO1 mRNA levels in wild type, rpb1-CTD11, cdk8Δ and rpb1-CTD11 cdk8Δ mutants before and after induction. In agreement with previous work, rpb1-CTD11 mutants had an impaired ability to activate INO1 expression upon induction (Figure 7A) [7], [45]. Upon deletion of CDK8, INO1 mRNA levels were robustly and reproducibly restored. This effect was corroborated with the suppression of the growth defect of CTD truncation mutants in media lacking inositol upon removal of CDK8 (Figure 7B). Consistent with this being a direct effect on mRNA synthesis, Rpb3 levels throughout the INO1 gene in rpb1-CTD11 mutants were significantly lower as compared to wild type. Furthermore, upon deletion of CDK8, the levels of RNAPII associated with the INO1 gene were restored (Figure 7C). While not statistically significant, we nevertheless observed a tendency for increased Rpb3 occupancy at the 3′ end of the gene in cdk8Δ and rpb1-CTD11 cdk8Δ mutants.


High-throughput genetic and gene expression analysis of the RNAPII-CTD reveals unexpected connections to SRB10/CDK8.

Aristizabal MJ, Negri GL, Benschop JJ, Holstege FC, Krogan NJ, Kobor MS - PLoS Genet. (2013)

INO1 expression and RNAPII association defects of rpb1-CTD11 mutants were suppressed by deleting CDK8.Cells were grown in inositol containing media (200 µM) to constitute the uninduced sample, and shifted to inositol deplete media for 4 hrs to constitute the induced sample. (A) qRT-PCR analysis of INO1 expression revealed a restoration of expression upon loss of CDK8. INO1 mRNA levels were normalized to ACT1 levels. (B) The sensitivity of CTD truncation mutants containing 11 or 12 repeats to growth in media lacking inositol was suppressed by deleting CDK8. (C) ChIP analysis of Rpb3 binding along the INO1 gene. Asterisks indicate induced conditions. Rpb3 enrichment along the INO1 gene was normalized to an intergenic region of chromosome V. Error bars represent standard deviations of values from three replicates.
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Related In: Results  -  Collection

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pgen-1003758-g007: INO1 expression and RNAPII association defects of rpb1-CTD11 mutants were suppressed by deleting CDK8.Cells were grown in inositol containing media (200 µM) to constitute the uninduced sample, and shifted to inositol deplete media for 4 hrs to constitute the induced sample. (A) qRT-PCR analysis of INO1 expression revealed a restoration of expression upon loss of CDK8. INO1 mRNA levels were normalized to ACT1 levels. (B) The sensitivity of CTD truncation mutants containing 11 or 12 repeats to growth in media lacking inositol was suppressed by deleting CDK8. (C) ChIP analysis of Rpb3 binding along the INO1 gene. Asterisks indicate induced conditions. Rpb3 enrichment along the INO1 gene was normalized to an intergenic region of chromosome V. Error bars represent standard deviations of values from three replicates.
Mentions: A previously characterized phenotype of CTD truncation mutants is reduced activation of INO1 and GAL10 upon switching to inducing conditions. Therefore, we investigated if loss of CDK8 could also suppress these expression defects of CTD truncation mutants [7]. Focusing on INO1, a gene important for the synthesis of inositol and survival in response to inositol starvation, we measured INO1 mRNA levels in wild type, rpb1-CTD11, cdk8Δ and rpb1-CTD11 cdk8Δ mutants before and after induction. In agreement with previous work, rpb1-CTD11 mutants had an impaired ability to activate INO1 expression upon induction (Figure 7A) [7], [45]. Upon deletion of CDK8, INO1 mRNA levels were robustly and reproducibly restored. This effect was corroborated with the suppression of the growth defect of CTD truncation mutants in media lacking inositol upon removal of CDK8 (Figure 7B). Consistent with this being a direct effect on mRNA synthesis, Rpb3 levels throughout the INO1 gene in rpb1-CTD11 mutants were significantly lower as compared to wild type. Furthermore, upon deletion of CDK8, the levels of RNAPII associated with the INO1 gene were restored (Figure 7C). While not statistically significant, we nevertheless observed a tendency for increased Rpb3 occupancy at the 3′ end of the gene in cdk8Δ and rpb1-CTD11 cdk8Δ mutants.

Bottom Line: Truncating the CTD altered RNAPII occupancy, leading to not only decreases, but also increases in mRNA levels.This suggested a positive role of Cdk8 in relationship to RNAPII, which contrasted with the observed negative role at the activated INO1 gene.Here, loss of CDK8 suppressed the reduced mRNA expression and RNAPII occupancy levels of CTD truncation mutants.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
The C-terminal domain (CTD) of RNA polymerase II (RNAPII) is composed of heptapeptide repeats, which play a key regulatory role in gene expression. Using genetic interaction, chromatin immunoprecipitation followed by microarrays (ChIP-on-chip) and mRNA expression analysis, we found that truncating the CTD resulted in distinct changes to cellular function. Truncating the CTD altered RNAPII occupancy, leading to not only decreases, but also increases in mRNA levels. The latter were largely mediated by promoter elements and in part were linked to the transcription factor Rpn4. The mediator subunit Cdk8 was enriched at promoters of these genes, and its removal not only restored normal mRNA and RNAPII occupancy levels, but also reduced the abnormally high cellular amounts of Rpn4. This suggested a positive role of Cdk8 in relationship to RNAPII, which contrasted with the observed negative role at the activated INO1 gene. Here, loss of CDK8 suppressed the reduced mRNA expression and RNAPII occupancy levels of CTD truncation mutants.

Show MeSH
Related in: MedlinePlus