MSCA Postdoctoral Fellowship - The Role of m6A Methylation in Response to Genotoxic Stress and in Cancer

The LRTOX laboratory of the French Authority for Nuclear Safety and Radioprotection (ASNR) welcomes applicants for a Marie S. Curie Action (MSCA) Postdoctoral Fellowship for the following research project:

*The Role of m6A Methylation in Response to Genotoxic Stress and in Cancer

Supervisor: Guillaume Varès

In response to ionizing radiation (IR) exposure, cells trigger a variety of complex and coordinated mechanisms, which include DNA Damage Response (DDR), the control of cellular proliferation or apoptosis induction. IR exposure might lead to genomic instability, chromosomal aberrations and cancer. However, while the effects of high doses of IR are well understood in a variety of models, including colon cancer [1], a wide variety of issues and questions remain concerning low dose effects, for which there is no consistent evidence yet of increased cancer incidence [2]. It is therefore crucial to better understand the molecular responses to low dose IR (LDR) in order to improve the assessment of cancer and non-cancer risks.

Gene regulation was previously thought to be driven mainly by the changes in transcription activation and repression under the control of transcription regulators. However, there is mounting evidence that post-transcriptional mechanisms, including the control of RNA translocation, splicing, translation, decay and stability, play a crucial role in shaping responses during physiological processes (development, immunity, stress responses, etc.). RNA stability regulation has been associated with the pathophysiology of various diseases [3–5]. This mechanism is especially important when rapid changes of gene expression are necessary, such as for controlling rhythmic gene expression in the circadian clock machinery [6].

In recent years, N6-methyladenosine (m6A) RNA modification has gained a lot of attention as a major regulatory marker controlling diverse aspects of RNA metabolism and function, including: alternative splicing, structure switching, export, miRNA maturation, stability, translation, m7G-independent translation and decay [7]. The presence and role of m6A on RNA are determined by the interplay between m6A-interacting proteins: methyltransferases (writers: METTL3, METTL14, etc.), demethylases (erasers: FTO, ALKBH5, etc.) and m6A-binding proteins (readers: YTHDF1, YTHDC2, etc.).

m6A methylation can regulate the expression of oncogenes and control cell differentiation, proliferation and migration, stem-like phenotype and tumor microenvironment [8]. Oncogenic properties of m6A regulating proteins have been reported in a number of cancer models. In colon cancer, METTL3 is over-expressed and promotes translation of EGFR and the Hippo pathway effector TAZ [9]. YTHDF1 is induced by c-Myc and is over-expressed in colon cancer, leading to cancer cell proliferation and resistance to anticancer drugs [10].

The detailed role of m6A modifications in radiation biology is still poorly known but is currently under intensive investigation [11]. m6A-related radiation-sensitive biomarkers (such as Ncoa4, Ate1 and Fgf22) exhibit persistent dose-dependent m6A methylation levels and may serve as biomarkers of radiation exposure [12]. m6A methylation may also be used to predict radiation sensitivity [13] or to develop new treatment strategies [14]. 

Recently, it was shown that it is possible to detect RNA modifications using long-read direct RNA sequencing by comparing an experimental test condition with a control condition containing no RNA modifications after knock-down of the METTL3 methyltransferase[15]. However, given the described effects of METTL3 on DDR and carcinogenesis, approaches relying on inhibiting its expression to identify m6A methylation sites may not be the most suitable for studies investigating time course of m6A regulation after stress or during carcinogenesis. Alternatively, it is possible to obtain a de novo profile of m6A sites by detecting increased mismatches around modified RNA bases [16] or by focusing on specific types of sequences through fraction modification values [17].

Here, we propose to investigate the dynamics and regulation of m6A methylation after exposure to low doses of ionizing radiation in a murine cell model through the following methodological and research aims:

1.To map RNA modifications transcriptome-wide using long-read sequencing (Nanopore technology) using a METTL3 shRNA-based approach [15] and an alternative de novo approach within filtered DRACH  motifs [17] and/or m6RIP-seq [18] + gene-specific m6A-RT-qPCR [19].  

2.To evaluate and characterize the role of m6A methylation in regulating the RNA stability and metabolism in colon carcinogenesis, using a model developed in our laboratory, that is, organoids derived from an APCf/f KRAS+/f CDX2-Cre-ERT2 (KPC:APC) inducible colon cancer model [20]. After tamoxifen administration KPC:APC mice develop tumors that mimic the histological and molecular characteristics of human colorectal cancer.

3.To measure m6A methylation modifications (and potentially other RNA methylation modifications) in the colonic tissues of the KPC:APC mouse exposed to low dose radiation exposure in relation to colon carcinogenesis and/or in cell lines after radiation exposure.

Besides an excellent access to infrastructure resources (histology, molecular biology, bioinformatics, etc.), the laboratory offers a supportive and collaborative environment, with multiple opportunities for personal growth.

If you wish to apply and submit a joint project, please send an email to guillaume.vares@asnr.fr, with your CV, cover letter and at least one recommendation letter.

The selected candidate and his/her supervisor will be responsible for writing a joint proposal on their project topic, which will be submitted to the Horizon Europe MSCA Postdoctoral Fellowship call and evaluated by experts appointed by the European Commission. Our internal application deadline is TBD, with the expectation that the selected candidate and his/her supervisor will complete a proposal by early September.

** Eligibility criteria:

- Candidates must be in possession of a doctoral degree before the call deadline (10 Sept. 2024) and have a maximum of 8 years full-time equivalent experience in research.

- “Mobility rule”: candidates must NOT have resided or carried out their main activity in France for more than 12 months in the 36 months immediately before the call deadline. Candidates can be from any nationality.