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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 1. E2F1 regulates RNA pol II phosphorylation and cap methylation. E2F1-ER expressed in rat fibroblasts was activated by addition of 100 nM 4-hydroxytamoxifen (+)or vehicle control (-), for 3 h. (A) RNA was extracted and RT-PCR performed with primers specific for CDC2 and GAPDH. (B) RNA was oligo-dT-purified (total transcripts, gray bars) and subsequently anti-m7G-purified (m7G transcripts, black bars) and RT-PCR performed with primers specific for CDC2. (C) E2F1-ER was activated by incubation in 100 nM 4-hydroxytamoxifen (OHT) for the time course indicated. Western blotting was used to detect total RNA pol II and Ser-5 phosphorylated RNA pol II in nuclear extracts.
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Figure 1: Figure 1. E2F1 regulates RNA pol II phosphorylation and cap methylation. E2F1-ER expressed in rat fibroblasts was activated by addition of 100 nM 4-hydroxytamoxifen (+)or vehicle control (-), for 3 h. (A) RNA was extracted and RT-PCR performed with primers specific for CDC2 and GAPDH. (B) RNA was oligo-dT-purified (total transcripts, gray bars) and subsequently anti-m7G-purified (m7G transcripts, black bars) and RT-PCR performed with primers specific for CDC2. (C) E2F1-ER was activated by incubation in 100 nM 4-hydroxytamoxifen (OHT) for the time course indicated. Western blotting was used to detect total RNA pol II and Ser-5 phosphorylated RNA pol II in nuclear extracts.

Mentions: In this study, we investigated regulation of methyl cap formation by E2F1 on its target transcript, the cyclin-dependent kinase, CDC2. E2F1 activity was regulated in fibroblasts by activation of E2F1-ER, a fusion protein of E2F1 and the estrogen receptor (ER).9 E2F1-ER is retained in the cytoplasm until addition of the ER ligand, 4-hydroxytamoxifen, promotes its movement to the nucleus, where it binds E2F1 recognition motifs proximal to transcription initiation sites.9 E2F1-ER was activated for 3 h; RNA was extracted, and RTPCR was used to demonstrate that the expression level of its target transcript, CDC2, was upregulated, whereas a control gene, GAPDH, was not (Fig. 1A). As had been observed previously, activation of E2F1 also resulted in an increase in the proportion of CDC2 transcripts with a methyl cap, as determined by anti-7-methylguanosine antibody immunoprecipitation followed by RTPCR (Fig. 1B). Methyl cap formation is dependent on recruitment of the methyl cap synthetic enzymes CE and RNMT to phosphorylated RNA pol II. Activation of E2F1 resulted in increased RNA pol II Ser-5 phosphorylation (Fig. 1C).


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

Aregger M, Cowling VH - Cell Cycle (2012)

Figure 1. E2F1 regulates RNA pol II phosphorylation and cap methylation. E2F1-ER expressed in rat fibroblasts was activated by addition of 100 nM 4-hydroxytamoxifen (+)or vehicle control (-), for 3 h. (A) RNA was extracted and RT-PCR performed with primers specific for CDC2 and GAPDH. (B) RNA was oligo-dT-purified (total transcripts, gray bars) and subsequently anti-m7G-purified (m7G transcripts, black bars) and RT-PCR performed with primers specific for CDC2. (C) E2F1-ER was activated by incubation in 100 nM 4-hydroxytamoxifen (OHT) for the time course indicated. Western blotting was used to detect total RNA pol II and Ser-5 phosphorylated RNA pol II in nuclear extracts.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3368866&req=5

Figure 1: Figure 1. E2F1 regulates RNA pol II phosphorylation and cap methylation. E2F1-ER expressed in rat fibroblasts was activated by addition of 100 nM 4-hydroxytamoxifen (+)or vehicle control (-), for 3 h. (A) RNA was extracted and RT-PCR performed with primers specific for CDC2 and GAPDH. (B) RNA was oligo-dT-purified (total transcripts, gray bars) and subsequently anti-m7G-purified (m7G transcripts, black bars) and RT-PCR performed with primers specific for CDC2. (C) E2F1-ER was activated by incubation in 100 nM 4-hydroxytamoxifen (OHT) for the time course indicated. Western blotting was used to detect total RNA pol II and Ser-5 phosphorylated RNA pol II in nuclear extracts.
Mentions: In this study, we investigated regulation of methyl cap formation by E2F1 on its target transcript, the cyclin-dependent kinase, CDC2. E2F1 activity was regulated in fibroblasts by activation of E2F1-ER, a fusion protein of E2F1 and the estrogen receptor (ER).9 E2F1-ER is retained in the cytoplasm until addition of the ER ligand, 4-hydroxytamoxifen, promotes its movement to the nucleus, where it binds E2F1 recognition motifs proximal to transcription initiation sites.9 E2F1-ER was activated for 3 h; RNA was extracted, and RTPCR was used to demonstrate that the expression level of its target transcript, CDC2, was upregulated, whereas a control gene, GAPDH, was not (Fig. 1A). As had been observed previously, activation of E2F1 also resulted in an increase in the proportion of CDC2 transcripts with a methyl cap, as determined by anti-7-methylguanosine antibody immunoprecipitation followed by RTPCR (Fig. 1B). Methyl cap formation is dependent on recruitment of the methyl cap synthetic enzymes CE and RNMT to phosphorylated RNA pol II. Activation of E2F1 resulted in increased RNA pol II Ser-5 phosphorylation (Fig. 1C).

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