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Mechanistic link between epitranscriptomics and a key driver of oncogenesis

par Ma, Haili
Président du jury Corazza, Francis
Promoteur Fuks, François
Publication Non publié, 2023-12-07

Thèse de doctorat

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Résumé :

Apart from modifications in DNA and histones, post-transcriptional RNA modifications are also being recognized as the third key aspect of epigenetics. The use of next-generation sequencing and improved mass spectrometry technology has revealed the presence of many previously unknown modifications in mRNA. This previously unknown field of RNA epigenetics is now referred to as epitranscriptomics. Recently, numerous new mRNA modifications were identified. N6-methyladenosine (m6A) is the most studied mRNA modification due to its high abundance. Recruitment of m6A binding proteins, known as “readers”, is crucial for controlling the fate of modified RNAs, including splicing, export, translation, and stability. Moreover, it plays a crucial role in tumor proliferation, differentiation, invasion, and metastasis. 5-methylcytosine (m5C), which is present in different types of RNAs, has not been as extensively studied. The role of m5C in mRNA processing and disease has not been fully explored yet, and “readers” of m5C have not been well identified.In this study, we used biotin pull-down followed by mass spectrometry approach to discover new readers of m5C-modified RNA. This led to a surprise: we found that the RNA-binding protein SRSF2 binds directly to m5C-marked RNAs. By characterizing the transcriptome-wide SRSF2 RNA-binding profile and m5C landscape in HeLa cells, we observe co-occurrence of the two features and notably a larger proportion of m5C-modified mRNAs associated with SRSF2 targets among the m5C sites with high stoichiometry. Depletion of m5C RNA methyltransferase NSUN2 reduces mRNA m5C levels and SRSF2 RNA-binding affinity and elicits alternative splicing effects similar to SRSF2 loss. Remarkably, we found that the SRSF2 proline 95 to histidine (P95H) mutation reduces the binding affinity of SRSF2 to m5C. Using SRSF2 structure, we identified residues involved in m5C recognition and the impact of SRSF2P95H mutation. Furthermore, transcriptome-wide binding analysis revealed that NSUN2 depletion on the one hand, and the SRSF2P95H mutation on the other, lead to altered SRSF2 RNA-binding, notably to key leukemia-associated transcripts in human leukemia cells. Finally, we confirmed that low NSUN2 expression is associated with mRNA hypomethylation in leukemia patients. Above all, we systematically found in four independent cohorts a reduction in NSUN2 and we discovered that, in patients carrying the SRSF2P95H mutation, a strong reduction in NSUN2 is associated with a poor prognosis.Taken together, our study reveals an unsuspected function of the SRSF2 protein as a reader of m5C on mRNA. Our data strongly suggest that impairment of this function can at least partly contribute to oncogenesis. By linking epitranscriptomics to a frequent leukemia-associated mutation, our study opens potential novel therapeutic avenues for hematological malignancies.