Objective 1 (achieved):
A) Methods: 2-3 months
I implemented the LC-MS method for the quantification of N6-methyladenosine (m6A) and other RNA modifications (Figure 1).
B) Investigation/Results: 12 months
i) Analysed VHL -/- 786O cancer cells subjected to metabolic conditions hypothesized to affect steady-state RNA methylation. We found that short-term metabolite deprivation modestly increases m6A and m1A levels in mRNAs, which we aim to elucidate in Objective 2.
ii) In collaboration with the Johnson laboratory (University of Cambridge), we analysed RNA methylation in human naïve and effector CD8+ T-cells upon treatment with the ‘immuno-metabolite’ S-2-hydroxyglutarate (S2HG). We found that differentiating T-cells undergo hypermethylation of m1A and m5C and hypomethylation of an unstudied RNA modification in non-coding RNAs, which motivated us to focus in non-coding RNAs (Objectives 2 and 3).
iii) Working with Dr. Miha Modic, a postdoc in the host laboratory, we found that RNA methylation is also dynamic in mESCs in non-coding RNAs at different stages of pluripotency.
iv) As proposed in the Contingency Plan and to achieve Objective 4, we are generating genetic models of methyltransferases/demethylases with the help of students in the host lab and in collaboration with the Kouzarides Lab (University of Cambridge). We have generated cell lines knocked out for FTO and putative methyltransferases, and triple knocked down for mettl3/mettl14/wtap.
Objective 2 (partially achieved):
A) Methods: 6-9 months
Together with PhD students in the host lab, we have implemented and optimised the miCLIP technique (Figure 2) for the transcriptome-wide profile of m6A and other RNA modifications with satisfactory coverage and specificity in coding and non-coding RNAs. This is a collaborative effort with the Kouzarides lab.
B) Investigation/Results: 12 months
We have performed miCLIP in i) VHL -/- 786O cells subjected to specific metabolic conditions, ii) human/mouse naïve and effector CD8+ T-cells treated or not with S-2HG, and iii) naive and primed mouse ESCs. Results: Ongoing - we are currently performing in-depth analyses of miCLIP data sets.
Objective 3 (partially achieved):
Investigation/Results: 12 months
i) Based on Objective 1, we hypothesized that RNA methylation-induced changes in RNA structure may be a mechanism by which non-coding RNAs regulate cell function(s).
Results: Using in vitro experiments and LC-MS/MS, we found that some RNA modifications affect the stability of particular non-coding RNAs, and are performing validation experiments. This result is the foundation for ongoing experiments (see expected results until the end).
ii) We are testing if and how RNA modifications are associated with differential protein synthesis. Ongoing
Objective 4 (ongoing):
We are testing if modulation of RNA methylation affects T-cell and mESC differentiation.
Communication and Dissemination of results:
The results of the four Objectives are likely to be published in impactful journals, and will warrant press dissemination to inform non-specialists of the impact of our research. I have presented this research to the wider scientific audience at the Francis Crick Institute, at the RNA UK meeting, and submitted an abstract to the upcoming EMBO workshop on “epitranscriptome in cell fate choice”.
I have taken part in teaching activities organized by UCL and the Francis Crick Institute. Specifically, I have presented my research in a engaging manner to six undergraduate UCL medical students in the SSC program to bring awareness and curiosity into biomedical research and my project, showed them the host laboratory and performed small demonstrations of lab equipment.
