The synthesis of the N7-methylguanosine cap at the 5’ end of pre-mRNA occurs co-transcriptionally and is catalysed by a series of enzymes including the N7 RNA methyltransferase (RNMT), which along with its recently discovered activating subunit RAM, methylates the cap. RAM, which contains an RNA binding domain, is required to promote RNMT activity both in vitro and in vivo. Although the biochemical function of RAM has been characterized, its biological relevance remains elusive to date. The addition of the cap moiety is a crucial event in gene expression as it affects several processes within the mRNAs life cycle including mRNA processing, stability and translation. In stem cells, every step of mRNA metabolism is tightly regulated to maintain the undifferentiated state, allowing the expression of pluripotency genes and the concomitant repression of the lineage-specific ones. Here, I describe a critical role for the mRNA cap methylation in the maintenance of pluripotency. RNMT and RAM are highly expressed in mESCs compared to differentiated cells. The reprogramming of MEFs to iPS totally restores the elevated expression levels of RNMT and RAM suggesting that high levels of the two proteins are a feature of pluripotent cells. Even more exciting, the same expression is conserved amongst species as also hESCs and hiPSCs exhibit high levels of RNMT and RAM compared to fibroblasts. So far, RNMT and RAM were described as a complex in all cells lines examined and it was assumed that are similarly regulated, instead surprisingly, during in vitro neural differentiation a specific reduction in RAM protein levels is observed. Gain- and loss-of-function studies have been employed to demonstrate that specifically high RAM levels are required for the maintenance of pluripotency. In fact, RAM depletion causes a major reduction in the methyl cap levels of important pluripotency factors, ultimately resulting in a decreased of the protein levels. Therefore, RAM is found to function as modulator of RNMT activity, whereby it promotes cap methylation of fundamental transcripts required for the maintenance of ESCs pluripotency. I have also found that during differentiation RAM is down regulated post-transcriptionally, and therefore current studies are focused on investigating the role of RAM phosphorylation at Serine-36, which correlates with proteosomal degradation. Together the data corroborate previous findings about the methyl cap formation being a critical and regulated process within gene expression and propose a novel implication of this modification in the maintenance of pluripotency.
|Date of Award||2015|
|Supervisor||Victoria Cowling (Supervisor)|