mRNA cap guanine-N7 methyltransferase (RNMT) catalyzes the S-adenosyl-dependent methylation of the 5’ cap on mRNA at the N-7 position of guanosine. RNA guanine-N7 methyltransferase activating subunit (RAM) allosterically binds to RNMT, which enhances its methyltransferase activity. RAM is phosphorylated at Ser36; however, how this post-translational modification impacts its interaction with RNMT is still unclear. Ser36 of RAM is positioned within a positively charged binding pocket of RNMT, indicating that phosphorylation would improve the binding affinity between these two proteins. Using protein semi-synthesis, we discovered that the first 45 amino acids of RAM is sufficient for full binding to RNMT, and that phosphorylation of Ser36 does increase the binding affinity around six-fold. / 2026-02-28T00:00:00Z

Yes / The mRNA cap stabilizes transcripts and recruits processing and translation factors. Grasso et al. report that the mRNA cap methyltransferase RNMTRAM is highly expressed in embryonic stem cells and is important for pluripotency-associated gene expression. Repression of RAM occurs during neural differentiation and is important for expression of neuralassociated genes. / Wellcome Trust

The N-7 methylguanosine cap structure is conserved from yeast to man. It is essential for cell proliferation as it influences several steps in eukaryotic gene expression including transcription, pre-mRNA processing, RNA export and translation. The N-7 methylguanosine cap is added co-transcriptionally to RNA pol II transcripts. In mammals, two enzymes catalyse the synthesis of the N7-methylguanosince cap. RNGTT adds an inverted guanosine group to the first transcribed nucleotide and RNMT methylates the guanosine cap at the N7-position. RNMT consists of a catalytic domain and an N-terminal domain that is absent in lower eukaryotes. Experiments presented in this thesis revealed that the N-terminus mediates RNMT recruitment to transcription start sites. Furthermore, it was found that the RNMT N-terminal domain is phosphorylated at Threonine-77 (T77) by CDK1/Cyclin B in a cell cycle-dependent manner during G2/M-phase. RNMT T77 phosphorylation activates cap methyltransferase activity in vitro. Furthermore, it negatively regulates the interaction of RNMT with KPNA2 (Importin-a), which was found to inhibit RNMT activity in vitro. RNMT T77 phosphorylation is required for normal cell proliferation suggesting an important biological function. Initial experiments indicated that RNMT T77 phosphorylation functions to regulate gene expression in a gene-specific manner. Future work is focused on establishing an experimental system to perform a genome-wide study in order to elucidate which transcripts are affected by RNMT T77 phosphorylation. To summarise, this study for the first time revealed that the RNA cap methyltransferase activity is regulated in a cell-cycle dependent manner.

The mRNA cap added to the 5’ end of nascent transcripts is required for the efficient gene expression in eukaryotes. In vertebrates, the guanosine cap is methylated at N7 position by RNMT, which is in complex with its activating subunit RAM. Additionally, the first and second transcribed nucleotides can be methylated at ribose O2 position by CMTR1 and CMTR2 respectively. The mRNA cap protects transcripts from degradation and recruits cap-binding factors to promote pre-mRNA processing, nuclear export and translation initiation. In mouse embryonic stem cells (mESCs), high levels of RAM maintain expression of pluripotency factors. Differentiation of mESCs to neural progenitors is accompanied by a suppression of RAM, resulting in downregulation of pluripotency factors and efficient formation of neural cells. Here, I demonstrated that the suppression of RAM during neural differentiation is promoted via ubiquitination and proteasomal degradation. Concurrently, neural differentiation is associated with an increase in CMTR1 expression, creating a developmental cap methyltransferase switch. Moreover, differentiation into endodermal and mesodermal lineages exhibited distinct changes in the mRNA capping enzymes expression. In mESCs, RAM promotes expression of translation-associated proteins and promotes global loading of mRNA on ribosomes. RAM contributes to the ESC-specific gene expression program, by maintaining optimal expression of pluripotency-associated transcripts and inhibiting expression of neural genes. Chromatin immunoprecipitation revealed that RAM, RNMT and CMTR1 promote binding of RNA polymerase II at gene loci. In RAM-repressed cells, RNA polymerase II binding was reduced at pluripotency-associated genes, but relatively increased at neural genes. Moreover, knock-down of RNMT or CMTR1 induced increased or decreased accumulation of RNA polymerase II at promoter proximal regions respectively. In naïve T cells, Rnmt or Cmtr1 conditional knock-outs caused downregulation of translation-related transcripts and upregulation of cell cycle transcripts. Furthermore, many transcripts were specifically dependent on RNMT or CMTR1 for expression, demonstrating distinct roles of these cap methyltransferases. Thus, the mRNA cap methylation emerges as an important regulator of pluripotency and differentiation, modulating gene expression at transcriptional and post-transcriptional levels.

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