Abstract
The mRNA cap consists of a series of co-transcriptional modifications on nascent transcripts that confer protection from degradation and mediate RNA processing and translation. The addition of an inverted guanosine cap to the 5’-end of the transcript and its subsequent methylation along with the methylation of the first and second transcribed nucleotides are catalysed by several enzymes to form a complete cap structure. Growing evidence suggests that the mRNA cap and the mRNA capping enzymes can be regulated in a context-dependent manner by different molecules and signalling pathways either through co-factors or post-translational modifications.Recent studies have demonstrated how cell-housekeeping processes such as mRNA splicing and protein synthesis regulation can have unexpected roles in cell-fate determination. The Cowling lab discovered that RNMT and RAM, which form the cap methyltransferase complex, are expressed at different levels in mouse primary tissues, and RAM is particularly highly expressed in mESCs. RAM is essential for pluripotency maintenance by controlling the expression of pluripotency-associated transcription factors in a positive feedback loop manner. During neural differentiation, RAM is degraded in an ERK1/2-dependent manner.
This work focused on further investigating the role of the mRNA capping enzymes in pluripotency and differentiation. CMTR1, the first nucleotide O-2 ribose methyltransferase, was found to be upregulated during embryoid body formation, LIF withdrawal and in vitro neural differentiation. The function of CMTR1 in mESCs and differentiation was explored by producing a Cmtr1∆S30-D31 cell line by CRISPR-Cas9, although it was not possible to obtain complete knockout clones, indicating that CMTR1 is essential for mESCs survival. Severe downregulation of CMTR1 resulted in a 40% decrease in first nucleotide ribose O-2 methylation, while global mRNA transcription was increased when compared to the control. Cmtr1∆S30-D31 mESCs displayed increased expression of pluripotency-associated genes. When induced to differentiate, these cells maintained high expression of pluripotency markers and displayed severe proliferation defects, suggesting that CMTR1 is important for the control of pluripotency and shift to differentiation. These effects were partially rescued. This study also indicated a novel nucleolar expression of CMTR1, even though its function is still to be elucidated. Finally, the expression of all known mRNA capping enzymes was found to be highly variable across adult mouse primary tissues, contributing to the concept that these enzymes are regulated in a context-dependent manner.
| Date of Award | 2020 |
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| Original language | English |
| Awarding Institution |
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| Sponsors | Wellcome Trust |
| Supervisor | Victoria Cowling (Supervisor) |