Specific regulation of mRNA cap methylation by the c-Myc and E2F1 transcription factors

Abstract

Methylation of the mRNA 5' guanosine cap is essential for efficient gene expression. The 5' methyl cap binds to eIF4E, which is the first step in the recruitment of mRNA to the 40S ribosomal subunit. To investigate whether mRNA cap methylation is regulated in a gene-specific manner, we established a method to detect the relative level of cap methylation on specific mRNAs. We found that two transcription factors, c-Myc and E2F1, induce cap methylation of their transcriptional target genes, and therefore, c-Myc and E2F1 upregulate gene expression by simultaneously inducing transcription and promoting translation. c-Myc-induced cap methylation is greater than transcriptional induction for the majority of its target genes, indicating that this is a major mechanism by which Myc regulates gene expression.

Figure 1. m7G-mRNA immunoprecipitation

A) In vitro transcribed mRNA synthesized with a 5′ guanosine cap (G-mRNA), or a 7-methylguanosine cap (m7G-mRNA) was offered as a substrate in an immunoprecipitation assay performed using polyclonal affinity-purified, anti-m7G antibodies and preimmune serum. The immunoprecipitated RNA and inputs were amplified by RTPCR. B) The immunoprecipitated mRNA detected in (A) was quantified, expressed as values relative to input material and normalised to the anti-m7G immunoprecipitation of G-mRNA. Values represent the mean of three experiments and error bars indicate the standard deviation. C) Immunoprecipitation of m7G-mRNA with anti-m7G antibodies was carried out in the presence of 2, 0.2 or 0.02 μM m7GTP or GTP, and bound RNA was detected as above. Experiment was performed three times with similar results.

Figure 2. c-Myc promotes transcription and mRNA cap methylation

A) c-Myc and Tubulin expression was detected by Western Blot in extracts from fibroblasts expressing c-MycWT (WT), c-MycΔMBII (Δ) or vector control (V). B) RNA was extracted and an anti-m7G immunoprecipitation was carried out. Input mRNA levels (grey bars), and m7G-mRNA levels (black bars) for the genes indicated were determined by RTPCR and normalised to vector control input mRNA and vector control m7G-mRNA, respectively. The immunoprecipitations were performed in duplicate and error bars indicate within experiment variation. The m7G-immunoprecipitations were performed at least twice on each of two independently isolated RNA samples with similar results.

Figure 3. c-Myc promotes increased polysome loading

A) Cell extracts from fibroblasts expressing c-Myc (black boxes) or vector control (white diamonds) were separated on sucrose gradients, fractions were collected, and the absorbance at 260 nM was measured for every other fraction and reported relative to input values. Experiment was performed five times with similar results. B) mRNA levels for the genes indicated were quantified by RTPCR for c-Myc-expressing (black bars) and vector control cells (gray bars), and were reported relative to input material. Experiment was performed on three sets of independently purified polysome preparations with similar results, and a representative set of data is shown in the figure. C) FBL and Nol5a mRNA (grey bars) and anti-m7G-mRNA (black bars) from fractions 26 and 32 was quantified by RTPCR. Experiment was performed three times with similar results.

Figure 4. E2F1 induces transcription and cap methylation

A) HA(E2F1) and Tubulin expression was detected by Western Blot in extracts from fibroblasts expressing E2F1-ER or vector control. B) E2F1-ER was activated with 4-hydroxytamoxifen for 0, 2 and 4 hrs. For the genes indicated, mRNA levels (grey bars) and m7G-mRNA levels (black bars) were determined by RTPCR and normalised to the 0 hr time point mRNA and m7G-mRNA, respectively. The immunoprecipitations were performed in duplicate and error bars indicate within experiment variation. The m7G-immunoprecipitation were performed at least twice on each of two independently isolated RNA samples with similar results.