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E2F1-dependent methyl cap formation requires RNA pol II phosphorylation.

Aregger M, Cowling VH - Cell Cycle (2012)

Bottom Line: The methyl cap is required for mRNA maturation, expression and stability.We demonstrate that E2F1 increases RNA pol II phosphorylation, which promotes recruitment of the methyl cap synthetic enzymes.Upregulation of RNA pol II phosphorylation is required for E2F1-dependent methyl cap formation.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee, UK.

ABSTRACT
Gene expression is a process integral to cell proliferation. The E2F family of transcription factors upregulates expression of transcripts whose products are essential for cell cycle progression. Here, we report that E2F1 promotes gene expression by an additional mechanism, that is, formation of the methyl cap on RNA pol II transcripts. The methyl cap is required for mRNA maturation, expression and stability. We demonstrate that E2F1 increases RNA pol II phosphorylation, which promotes recruitment of the methyl cap synthetic enzymes. Upregulation of RNA pol II phosphorylation is required for E2F1-dependent methyl cap formation.

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Figure 2. E2F1-dependent cap methylation requires RNA pol II phosphorylation. (A) Rat fibroblasts were incubated with 175 nM Actinomycin D (ActD), 5 μM DRB or vehicle control (-), for 30 min prior to addition of 10 μci/ml 3H uridine for 15 min. RNA was harvested, oligo-dT-purified and 3H uridine incorporation determined. (B) Following treatment with Act D or DRB, E2F1-ER was activated by addition of 100 nM 4-hydroxytamoxifen for 3 h (OHT). RNA was extracted, oligo-dT-purified and RT-PCR performed with primers specific for CDC2. (C) As in (b), RNA was oligo-dT-purified (total transcripts, gray bars) and anti-m7G-purified (m7G transcripts, black bars) and RT-PCR performed with primers specific for CDC2. Charts represent the average of three independent experiments and error bars indicate the standard deviation.
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Figure 2: Figure 2. E2F1-dependent cap methylation requires RNA pol II phosphorylation. (A) Rat fibroblasts were incubated with 175 nM Actinomycin D (ActD), 5 μM DRB or vehicle control (-), for 30 min prior to addition of 10 μci/ml 3H uridine for 15 min. RNA was harvested, oligo-dT-purified and 3H uridine incorporation determined. (B) Following treatment with Act D or DRB, E2F1-ER was activated by addition of 100 nM 4-hydroxytamoxifen for 3 h (OHT). RNA was extracted, oligo-dT-purified and RT-PCR performed with primers specific for CDC2. (C) As in (b), RNA was oligo-dT-purified (total transcripts, gray bars) and anti-m7G-purified (m7G transcripts, black bars) and RT-PCR performed with primers specific for CDC2. Charts represent the average of three independent experiments and error bars indicate the standard deviation.

Mentions: In order to determine the role of RNA pol II phosphorylation and transcription in the mechanism of methyl cap formation, cells were incubated with two inhibitors, Actinomycin D, a compound that forms a complex with DNA preventing movement of RNA polymerase, and DRB (Dichloro-1-β-D-ribofuranosylbenzimidazole riboside), an adenosine analog which inhibits RNA pol II kinases and, therefore, RNA pol II phosphorylation. The rate of RNA pol II transcription was determined by measuring the rate of 3H-uridine incorporation into oligo-dT-purified RNA (predominantly mRNA). Incubating cells for 30 min with 175 nM Actinomycin D or 5 μM DRB inhibited RNA pol II-dependent transcription by approximately 90% (Fig. 2A). Following treatment with DRB or Actinomycin D, CDC2 transcripts were depleted by approximately 50%, and CDC2 transcript levels became unresponsive to E2F1 (Fig. 2B).


E2F1-dependent methyl cap formation requires RNA pol II phosphorylation.

Aregger M, Cowling VH - Cell Cycle (2012)

Figure 2. E2F1-dependent cap methylation requires RNA pol II phosphorylation. (A) Rat fibroblasts were incubated with 175 nM Actinomycin D (ActD), 5 μM DRB or vehicle control (-), for 30 min prior to addition of 10 μci/ml 3H uridine for 15 min. RNA was harvested, oligo-dT-purified and 3H uridine incorporation determined. (B) Following treatment with Act D or DRB, E2F1-ER was activated by addition of 100 nM 4-hydroxytamoxifen for 3 h (OHT). RNA was extracted, oligo-dT-purified and RT-PCR performed with primers specific for CDC2. (C) As in (b), RNA was oligo-dT-purified (total transcripts, gray bars) and anti-m7G-purified (m7G transcripts, black bars) and RT-PCR performed with primers specific for CDC2. Charts represent the average of three independent experiments and error bars indicate the standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Figure 2. E2F1-dependent cap methylation requires RNA pol II phosphorylation. (A) Rat fibroblasts were incubated with 175 nM Actinomycin D (ActD), 5 μM DRB or vehicle control (-), for 30 min prior to addition of 10 μci/ml 3H uridine for 15 min. RNA was harvested, oligo-dT-purified and 3H uridine incorporation determined. (B) Following treatment with Act D or DRB, E2F1-ER was activated by addition of 100 nM 4-hydroxytamoxifen for 3 h (OHT). RNA was extracted, oligo-dT-purified and RT-PCR performed with primers specific for CDC2. (C) As in (b), RNA was oligo-dT-purified (total transcripts, gray bars) and anti-m7G-purified (m7G transcripts, black bars) and RT-PCR performed with primers specific for CDC2. Charts represent the average of three independent experiments and error bars indicate the standard deviation.
Mentions: In order to determine the role of RNA pol II phosphorylation and transcription in the mechanism of methyl cap formation, cells were incubated with two inhibitors, Actinomycin D, a compound that forms a complex with DNA preventing movement of RNA polymerase, and DRB (Dichloro-1-β-D-ribofuranosylbenzimidazole riboside), an adenosine analog which inhibits RNA pol II kinases and, therefore, RNA pol II phosphorylation. The rate of RNA pol II transcription was determined by measuring the rate of 3H-uridine incorporation into oligo-dT-purified RNA (predominantly mRNA). Incubating cells for 30 min with 175 nM Actinomycin D or 5 μM DRB inhibited RNA pol II-dependent transcription by approximately 90% (Fig. 2A). Following treatment with DRB or Actinomycin D, CDC2 transcripts were depleted by approximately 50%, and CDC2 transcript levels became unresponsive to E2F1 (Fig. 2B).

Bottom Line: The methyl cap is required for mRNA maturation, expression and stability.We demonstrate that E2F1 increases RNA pol II phosphorylation, which promotes recruitment of the methyl cap synthetic enzymes.Upregulation of RNA pol II phosphorylation is required for E2F1-dependent methyl cap formation.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee, UK.

ABSTRACT
Gene expression is a process integral to cell proliferation. The E2F family of transcription factors upregulates expression of transcripts whose products are essential for cell cycle progression. Here, we report that E2F1 promotes gene expression by an additional mechanism, that is, formation of the methyl cap on RNA pol II transcripts. The methyl cap is required for mRNA maturation, expression and stability. We demonstrate that E2F1 increases RNA pol II phosphorylation, which promotes recruitment of the methyl cap synthetic enzymes. Upregulation of RNA pol II phosphorylation is required for E2F1-dependent methyl cap formation.

Show MeSH
Related in: MedlinePlus