Investigating the synthesis, function and therapeutic potential of the mRNA methyl cap
We investigate the regulation and function of the methyl cap, and use this information to develop new therapies targeted at inhibiting tumour cell and parasite growth and proliferation.
The methyl cap is a structure found at the 5’ end of transcripts, which marks pre-mRNA for processing and translation initiation. Although it was discovered in the 1970s and characterised in the following years, function studies have been scarce in the last few decades and there are many unanswered questions about methyl cap function and regulation in the context of our current understanding of gene expression.
Figure 1 Methyl guanosine cap
c-Myc oncogene function requires cap methylation
Our interest in the methyl cap began whilst investigating the Myc family of proto-oncogenes, which are deregulated to some extent in most human cancers. Since therapies do not currently exist to inhibit Myc protein function in tumours, there is significant interest in their mode of action. We discovered that Myc proteins upregulate the proportion of transcripts with a “cap-like” structure, correlating with their increased translation. This was a surprising result since cellular regulation of methyl cap synthesis had not been recognised as a mechanism of gene regulation. We demonstrated that at least two mechanisms are required for Myc-induced methyl cap formation: 1) recruitment of TFIIH to promoters, which upregulates RNA pol II phosphorylation and recruits the capping enzymes, and 2) upregulation of SAHH enzyme which removes the inhibitory product of cap methylation. Crucially, we demonstrated that cap methylation is necessary for c-Myc-dependent gene expression, cell proliferation and cell transformation.
Figure 2 Mechanism of cap methylation
Discovery of an essential activator of cap methylation
During our research into c-Myc-regulated methyl cap synthesis, it became apparent that the mechanistic description of cap methylation is limited in mammalian cells. Since we are investigating the therapeutic potential of targeting methyl cap synthesis, it is critical to determine the mechanism involved. By performing mass spectrometric and biochemical analyses of cellular cap methyltransferase complexes, we determined that cellular RNMT is not a monomer as had been described, but rather exists in a complex with Fam103a1, a 12kDa uncharacterised protein. We demonstrated that Fam103a1 is required for recombinant and cellular RNMT cap methyltransferase activity, and for synthesis of the methyl cap on endogenous cellular transcripts, and therefore we describe Fam103a1 as the “obligate activator of the human cap methyltransferase”. We renamed Fam103a1 RAM, or RNMT-activating mini-protein. As expected of factor required for cellular cap methylation, RAM is indirectly required for transcript stability, translation, cell viability and cell proliferation.
Figure 3 RAM-RNMT
- Aregger, M. and Cowling, V. H. (2013). Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites. Biochem J 455, 67-73
- Cowling, V. H., Turner, S. A. and Cole, M. D. (2013). Burkitt's lymphoma-associated c-Myc mutations converge on a dramatically altered target gene response and implicate Nol5a/Nop56 in oncogenesis. Oncogene
- Aregger, M. and Cowling, V. H. (2012). E2F1-dependent methyl cap formation requires RNA pol II phosphorylation. Cell cycle 11, 2146-2148
- Gonatopoulos-Pournatzis, T., Dunn, S., Bounds, R. and Cowling, V. H. (2011). RAM/Fam103a1 is required for mRNA cap methylation. Mol Cell 44, 585-596
- Cowling, V. H. (2010). Enhanced mRNA cap methylation increases cyclin D1 expression and promotes cell transformation. Oncogene 29, 930-936
- Cole, M. D. and Cowling, V. H. (2009). Specific regulation of mRNA cap methylation by the c-Myc and E2F1 transcription factors. Oncogene 28, 1169-1175
- Fernandez-Sanchez, M. E., Gonatopoulos-Pournatzis, T., Preston, G., Lawlor, M. A. and Cowling, V. H. (2009). S-adenosyl homocysteine hydrolase is required for Myc-induced mRNA cap methylation, protein synthesis, and cell proliferation. Mol Cell Biol 29, 6182-6191