We investigate the synthesis, function and therapeutic potential of the mRNA methyl cap in mammals.
An introduction to the mRNA methyl cap
Global and gene-specific regulation of gene expression is integral to most cellular events, from the basic, e.g. the cell cycle and apoptosis, to the specialised, e.g. immune cell and neuronal function. Cellular proteins are largely synthesised from mRNA, and transcripts undergo several essential processing events prior to their maturation into translation-competent mRNA. A structure found at the 5’ end of transcripts, known as the “methyl cap”, 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.
- Cowling VH (2009) Regulation of mRNA cap methylation Biochemical Journal 2009, Biochem J. 2009 Dec 23;425(2):295-302. doi:10.1042/BJ20091352
- Cole MD and Cowling VH (2008) Transcription-independent functions of MYC: regulation of translation and DNA replication Nature Reviews Mol Cel Biol 2008 Oct; 9 (10):810-5. Epub 2008 Aug 13. doi:10.1038/nrm2467
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.
- Fernandez-Sanchez ME, Gonatopoulos-Pournatzis T, Preston G, Lawlor MA, and Cowling VH (2009) S-Adenosyl Homocysteine Hydrolase (SAHH) is required for Myc-induced mRNA cap methylation, protein synthesis and cell proliferation Mol Cell Biol 2009, Dec;29(23):6182-91. Epub 2009 Oct 5. doi:10.1128/MCB.00973-09
- Cowling VH (2010) Enhanced mRNA cap methylation increases Cyclin D1 expression and promotes cell transformation Oncogene. 2010 Feb 11;29(6):930-6. Epub 2009 Nov 16. doi:10.1177/1947601910378025
- Cole MD and Cowling VH (2009) Specific regulation of mRNA cap methylation by the c-Myc and E2F1 transcription factors Oncogene 2009 Mar 5;28(9):1169-75. Epub 2009 Jan 12. doi:10.1038/onc.2008.463
- Cowling VH and Cole MD (2007) The Myc Transactivation Domain Promotes Global Phosphorylation of the RNA pol II Carboxy-terminal Domain Independently of Direct DNA Binding Mol Cell Biol, 2007 March; 27(6) p2059-2073 doi:10.1128/MCB.01828-06
Discovery of RAM: RNMT activating miniprotein
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.
- Gonatopoulos-Pournatzis T, Dunn S, Bounds R, and Cowling VH (2011) RAM/Fam103a1 is required for mRNA cap methylation Molecular Cell 2011 Nov18;44(4):585-596 doi:10.1016/j.molcel.2011.08.041