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Metabolic control of RNA-protein and RNA-RNA interactions in cellular transformation.

Periodic Reporting for period 1 - M6Abolic-RNA-duplex (Metabolic control of RNA-protein and RNA-RNA interactions in cellular transformation.)

Reporting period: 2016-09-01 to 2018-08-31

What is the problem being addressed?
In this project, we proposed to study RNA methylation as a ‘dynamic’ and ‘regulatory mechanism’ of RNA structure, RNA-protein assembly and, therefore, RNA function. It is now known that N6-methyladenosine is a RNA modification with key roles in cell differentiation and development. Yet, there is limited insight on the 'upstream' regulatory mechanisms of m6A marks that lead to cell fate transitions, and whether other RNA modifications are regulated in a similar fashion is unexplored. In this context, this research was designed to approach the ‘metabolic dependencies’ of RNA methylation in order to elucidate the ‘dynamics’ and ‘function(s)’ of RNA methylation in cell differentiation and proliferation. This project has been focused in 786O cancer cells and two models of cell differentiation: CD8+ T-cells and mouse embryonic stem cells (mESCs).

Why is it important for society?
At the completion stage, this project is expected to provide far-reaching insights on fundamental regulatory mechanisms of RNA function, and it may lead to the development of new methodologies in:
i) Regenerative medicine, aimed at the control of cellular pluripotency/differentiation;
ii) Immunotherapy, aimed at the efficacy of T-cell differentiation into central/effector memory T-cells, and/or metabolic treatments in T-cell homeostasis;
iii) Cancer cell biology, aimed at targeting RNA methylation

What are the overall objectives?
(Initial objectives were updated as below due to project advancement and methodologies developed in the host lab)
Objective 1: Metabolic and cell differentiation states associated with ‘hyper’- or ‘hypo’-methylated RNAs - achieved
Objective 2: Transcriptome-wide profile of RNA methylation in cell differentiation and metabolic states associated with ‘hyper’ or ‘hypo’ methylation – partially achieved
Objective 3: Elucidate the role of RNA methylation in the structure and function of ‘dynamic’ RNAs (objective 1) – partially achieved
Objective 4: Significance of RNA methylation in cell fate transitions and cancer cell proliferation – ongoing
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.
A- We have expanded the scientific scope of our research by studying m6A, as initially proposed in Objective 1, as well as m1A and another RNA modification whose function(s) are largely unknown.
B- Objective 3 represents a relevant effort towards elucidating the mechanistic role of RNA methylation, which we did not propose initially.
C- We have extended the biological impact of our research by studying RNA methylation in two models of cell differentiation (CD8+ T-cells and embryonic stem cells) and in a clinically relevant cancer cell model: VHL -/- and VHL +/+ 786O cells.

Expected results until the end of the project.
Objective 2 will reveal the methylation signatures of cell differentiation and metabolic regulation, which we will test for mechanistic (Objective 3) and biological role (Objective 4). For Objectives 3, we expect the in vitro and transfection experiments with hyper/hypo-methylated RNAs to reveal how dynamic RNA methylation regulates RNA function and protein synthesis.
Upon completion, the expected results are likely to reveal novel functions of RNA methylation, and non-canonical roles of non-coding RNAs in gene regulation. These results may provide proof-of-concepts to target the RNA epitranscriptome in biomedicine.
Elucidating the dynamics, regulation and function(s) of RNA methylation in these cell systems may lead to i) new strategies to control cell pluripotency/differentiation and/or ii) modulation of RNA structure/function for biomedical-biotechnological applications.
LC-MS/MS Quantification of RNA methylation
miCLIP for the transcriptome-wide profile of RNA methylation