AbstractThe 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.
|Date of Award||2017|
|Supervisor||Victoria Cowling (Supervisor)|
- mRNA cap
- mRNA capping
- Embryonic stem cell
- Neural differentiation
- RNA polymerase II
- T cells