E2F1-dependent methyl cap formation requires RNA pol II phosphorylation

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.

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.

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.