The Myc proteins c-Myc and N-Myc are essential for development and tissue homoeostasis. They are up-regulated by growth factors and transmit the signal for cell growth and proliferation. Myc proteins are also prominent oncogenes in many human tumour types. Myc proteins regulate the transcription of protein-encoding mRNAs and the tRNAs and rRNA which mediate mRNA translation into protein. Myc proteins also up-regulate translation by increasing addition of the 7-methylguanosine cap (methyl cap) to the 5′ end of pre-mRNA. Addition of the methyl cap increases the rate at which transcripts are translated by directing RNA modifications and translation initiation. Myc induces methyl cap formation by promoting RNA polymerase II phosphorylation which recruits the capping enzymes to RNA, and by up-regulating the enzyme SAHH (S-adenosylhomocysteine hydrolase), which neutralizes the inhibitory by-product of methylation reactions. Myc-induced cap methylation is a major effect of Myc function, being necessary for activated protein synthesis, cell proliferation and cell transformation. Inhibition of cap methylation is synthetic lethal with elevated Myc protein expression, which indicates the potential for cap methylation to be a therapeutic target.
- cell proliferation
- gene expression
- 7-methylguanosine cap
- mRNA translation
The Myc family genes c-myc and N-myc are essential genes which regulate basic cellular functions including protein synthesis, cell-cycle progression, various metabolic pathways and biosynthetic processes, and cell adhesion [1,2]. They are up-regulated in response to growth factors and co-ordinate the mechanisms which mediate the cell growth and proliferation response. Certain stem cell populations are dependent on Myc proteins to inhibit terminal differentiation and promote self-renewal . Deregulated Myc protein expression is a common feature of many human tumours, which results in cell proliferation becoming somewhat growth-factor-independent [3,4].
Myc proteins are bHLH-LZ (basic helix–loop–helix–leucine zipper) proteins which heterodimerize with another bHLH-LZ protein, Max . Myc–Max dimers bind to chromatin predominantly at transcription initiation sites, recruiting enzymic co-factors which mediate changes in gene expression [5–7]. Numerous cofactors have been isolated in Myc complexes, most of which are chromatin-modifying enzymes and components of the transcriptional machinery. Initially, Myc proteins were identified as (up and down) regulators of mRNA expression, which is transcribed by RNA pol (polymerase) II [2,5]. Subsequently, Myc proteins were found to regulate miRNAs (microRNAs) which are also RNA pol II transcripts, and to regulate RNA pol I and III, which transcribe tRNAs and rRNA [7,8]. Therefore Myc co-ordinately regulates protein synthesis by regulating the transcription of protein-encoding mRNAs, the miRNAs that controls their expression, and the rRNA and tRNAs required to translate the message into protein. The co-ordinated regulation of RNA pol I, II and III is an impressive feat; however, there is another essential component to Myc regulation of gene expression. RNA pol II transcripts must be modified at the 5′-end by addition of the 7-methylguanosine cap (the methyl cap) to become competent for translation and Myc promotes this step [9–11]. The present review describes the mechanism of Myc-regulated methyl cap formation, and its biochemical and biological significance.
The methyl cap
The methyl cap is a 7-methylguanosine group joined to the first nucleotide of RNA pol II transcripts [11,12]. Nascent RNA is transcribed with a triphosphate group at the 5′-end, designated ppp(5′)N, where p is a phosphate group and N is the first transcribed nucleotide. The 5′-hydroxy group of 7-methylguanosine is joined through a triphosphate linkage to the transcript, creating a 5′–5′ linkage, designated m7G(5′)ppp(5′)N, where m7G is 7-methylguanosine. This 5′–5′ linkage is unusual and in contrast with the 3′–5′ phosphodiester bond which links nucleotides in transcribed RNA. The 5′–5′ linkage facilitates specific recognition of the cap by the cap methyltransferase and also protects against attack by exonucleases.
1. An RNA 5′-triphosphatase catalyses removal of the terminal phosphate of nascent RNA ppp(5′)N to create the intermediate pp(5′)N.
2. A guanylyltransferase catalyses addition of GMP to the nascent transcript to create the cap G(5′)ppp(5′)N.
3. A guanine 7-methyltransferase catalyses methylation of the cap at the N-7 position to create the methyl cap m7G(5′)ppp(5′)N. SAM (S-adenosylmethionine) is the methyl donor.
The enzymes which catalyse formation of the methyl cap are essential for the viability of mammalian and yeast cells . In mammals, the first two steps are catalysed by a single polypeptide with two active sites, called CE (capping enzyme) or RNGTT (RNA guanylyltransferase and 5′-triphosphatase), and the third step is catalysed by a distinct cap methyltransferase enzyme called RNMT (RNA guanine 7-methyltransferase) . RNMT only catalyses methylation of guanosine in a cap structure and therefore the cap must form before the guanosine is methylated. Since the capping enzyme and cap methyltransferase enzymes are distinct, they have the potential to be differentially regulated.
mRNA methyl cap formation is described as occurring ‘co-transcriptionally’, i.e. while the RNA is being transcribed by RNA pol II . The methyl cap is only found on RNA pol II gene products and this is thought to be mediated by the unique CTD (C-terminal domain) of the large subunit of RNA pol II, which consists of approx. 50 repeats of a 7-amino-acid sequence (heptad) . The CTD is phosphorylated on different residues during the transcription cycle, and this is thought to govern the differential recruitment of RNA-modifying enzymes and factors, including the capping enzyme RNGTT and the cap methyltransferase RNMT. TFIIH (transcription factor IIH) phosphorylates the CTD on Ser5 of the heptad at the initial stages of transcription and this promotes recruitment of RNGTT and RNMT at the correct time and place to catalyse methyl cap formation [14,15].
The methyl cap is required for the translation of most mRNAs . There are many processes that the methyl cap has the potential to influence, including transcription, splicing, nuclear export of RNA, protection of RNA against exonuclease attack and initiation of translation. The latter is the process most comprehensively linked to the methyl cap. eIF (eukaryotic translation initiation factor) 4F binds to the methyl cap via eIF4E and promotes recruitment of the 40S ribosomal subunit to initiate translation . However, the precise role of the methyl cap on most endogenous mammalian transcripts is not known. It may be that the methyl cap has different biochemical role(s) on different transcripts.
Myc regulates formation of the methyl cap
The Myc proteins c-Myc and N-Myc (referred to as Myc henceforth), were found to regulate formation of the methyl cap on certain transcripts [9,10]. The increase in methyl cap levels correlated with increased translation and protein expression of the genes in question. Formation of the methyl cap was observed to be regulated by Myc on a significant number of genes, including most transcriptional target genes and some genes not transcriptionally regulated by Myc. It is important to note that induced formation of the methyl cap is not simply a result of the Myc transcriptional programme since Myc mutants that are completely defective for transcriptional activity remain competent to regulate methyl cap formation, albeit with reduced efficiency compared with the wild-type protein .
Myc binds to components of the general transcription factor TFIIH, and increases its recruitment to transcription initiation sites, correlating with increased RNA pol II phosphorylation and methyl cap formation (Figure 2) . Myc induces global changes in chromatin structure and this may also favour increased recruitment of the methyl cap-forming enzymes . As described below, although recruitment of TFIIH is required, it is not sufficient for Myc-regulated methyl cap formation .
Role of Myc-dependent cap methylation
What is the biological significance of Myc-induced methyl cap formation? As described above, an increase in the proportion of methyl-capped transcripts has been observed on all transcriptional targets of Myc and on other transcripts and is therefore likely to have a significant biological impact [9,10]. This was proved following the discovery of a second pathway in addition to TFIIH recruitment, required for Myc to activate cap methylation.
Cellular methylation reactions utilize the methyl donor SAM and produce a by-product, SAH (S-adenosylhomocysteine) . SAH has the potential to inhibit methylation reactions by competing with SAM for the methyltransferase active site. SAH can be hydrolysed and thus neutralized in cells by the enzyme SAHH (S-adenosyl homocysteine hydrolase). Elevating Myc levels produces inhibitory concentrations of SAH, which must be removed in order for Myc-induced cap methylation to proceed (Figure 2) . Myc increases expression of SAHH enzyme both by regulating its transcription and cap methylation. Elevation of SAHH expression removes excess SAH and permits Myc-induced cap methylation. Myc also regulates methylation of other RNAs, DNA and proteins; however, when these methylations were investigated, they were not found to be dependent on up-regulation of SAHH . Either the cap methyltransferase may experience higher local concentrations of SAH than the other methyltransferases, or the cap methyltransferase enzyme may be more sensitive to SAH levels.
The discovery of SAHH allowed the question of the biological significance of Myc-induced methyl cap formation to be addressed. Inhibition of SAHH expression specifically inhibited cap methylation, whereas Myc-induced transcription was unaffected . Loss of Myc-induced cap methylation resulted in loss of Myc-induced protein synthesis, cell proliferation and cell transformation, thus establishing that Myc-induced cap methylation is a biologically significant Myc effector. This was confirmed by use of the methyltransferase inhibitor, tubercidin, which functions by inhibiting SAHH. Similarly to inhibition of SAHH expression, a specific concentration of tubercidin was observed to inhibit Myc-induced cap methylation, but not transcription . This concentration of tubercidin was also found to inhibit Myc-induced protein synthesis, cell proliferation and cell transformation. Interestingly, although tubercidin inhibited basic cellular processes in cells with elevated Myc expression, it had no effect on cells with endogenous levels of Myc, indicating that cap methylation may be an important therapeutic target in anticancer therapies.
Cap methylation has been established as a major effector of Myc function. Research is now focused on investigating the precise biochemical role of Myc-induced cap methylation. Following the finding that inhibition of cap methylation is synthetic lethal with elevated Myc expression, investigating the role of cap methylation in animal models of carcinogenesis is a priority.
V.H.C. holds a Medical Research Council Career Development Award. Recent work described in this review was funded by the Medical Research Council, Cancer Research UK, Wellcome Trust, Tenovus Scotland and the Biotechnology and Biological Sciences Research Council.
Post-Transcriptional Control: mRNA Translation, Localization and Turnover: A Biochemical Society Focused Meeting held at University of Edinburgh, U.K., 8–10 June 2010. Organized and Edited by Matthew Brook (Edinburgh, U.K.), Mark Coldwell (Southampton, U.K.), Simon Morley (Sussex, U.K.) and Nicola Gray (Edinburgh, U.K.).
Abbreviations: bHLH-LZ, basic helix–loop–helix–leucine zipper; CTD, C-terminal domain; eIF, eukaryotic initiation factor; miRNA, microRNA; pol, polymerase; RNGTT, RNA guanylyltransferase and 5′ triphosphatase; RNMT, RNA guanine 7-methyltransferase; SAH, S-adenosylhomocysteine; SAHH, S-adenosylhomocysteine hydrolase; SAM, S-adenosylmethionine; TFIIH, transcription factor IIH
- © The Authors Journal compilation © 2010 Biochemical Society