Myc Regulation of mRNA Cap Methylation

Abstract

The c-myc proto-oncogene regulates the expression of 15% to 20% of all genes, depending on the cell type, and the regulation is usually modest (1.5- to 2.0-fold). The authors discovered that in addition to regulating mRNA abundance, c-Myc regulates the formation of the 7-methylguanosine cap on many mRNAs, including transcriptional target genes and others not transcriptionally activated. Because the 7-methylguanosine cap is required for effective translation, enhanced methyl cap formation leads to increased protein production from Myc-responsive genes that exceeds the transcriptional induction. Increased cap methylation is linked to Myc-dependent enhanced activity of 2 critical kinases, TFIIH and p-TEFb, which phosphorylate the RNA polymerase II carboxy-terminal domain (CTD). Phosphorylation of the CTD recruits RNGTT and RNMT, the enzymes involved in mRNA capping, to the nascent transcript. Evidence is accumulating that enhanced cap methylation makes a significant contribution to Myc-dependent gene regulation and protein production.

Figure 1.

Enzymatic steps of mRNA capping. The primary mRNA transcript is synthesized with a 5′ triphosphate (pppN). The enzyme RNGTT (CE, or capping enzyme) acts as a phosphatase to remove the terminal phosphate (ppN). The RNGTT enzyme then uses GTP to create a G5′ppp5′N cap. This cap structure is subsequently recognized by a second enzyme RNMT to methylate the terminal guanosine.

Figure 2.

mRNA cap formation is coupled to phosphorylation of the RNA polymerase II carboxy-terminal domain (CTD). RNA polymerase II forms a preinitiation complex at the promoter with an unphosphorylated CTD (Initiation). The TFIIH basal transcription factor is recruited to phosphorylate the CTD on serine-5 (paused polymerase). Myc can enhance the level and recruitment of TFIIH to promoters. The phosphorylated CTD recruits the first capping enzyme (RNGTT) to add the reverse guanosine to the 5′ end of the mRNA (guanylylation). The cap methylation enzyme (RNMT) is also recruited by the phospho-CTD to methylate the GpppN-cap to yield m7G-pppN (methylation). Only the methylated cap is recruited by eIF4E in the cytoplasm to load efficiently onto ribosomes for translation.

Figure 3.

Enhanced mRNA cap methylation makes a significant contribution to the Myc response. Data are presented for an average of 8 Myc target genes from a previous study. Myc-null fibroblasts were reconstituted with exogenous Myc, and total mRNA levels were measured by RT-PCR. mRNAs were also selected with antibodies for the presence of a m7G cap. In comparison to the parental cells, Myc induced a 1.8-fold average increase in total mRNA abundance. However, the average increase in m7G-containing mRNAs was 4.9-fold, which reflects mRNA abundance and an increased fraction of mRNA with a methylated cap. Because m7G-containing mRNAs are translated much more efficiently than mRNAs with a nonmethylated cap, the net increase in protein levels are more reflective of the m7G-induction.