SRSF2 plays an unexpected role as reader of m⁵C on mRNA, linking epitranscriptomics to cancer

A common mRNA modification is 5-methylcytosine (m⁵C), whose role in gene-transcript processing and cancer remains unclear. Here, we identify serine/arginine-rich splicing factor 2 (SRSF2) as a reader of m⁵C and impaired SRSF2 m⁵C binding as a potential contributor to leukemogenesis. Structurally, we identify residues involved in m⁵C recognition and the impact of the prevalent leukemia-associated mutation SRSF2^P95H. We show that SRSF2 binding and m⁵C colocalize within transcripts. Furthermore, knocking down the m⁵C writer NSUN2 decreases mRNA m⁵C, reduces SRSF2 binding, and alters RNA splicing. We also show that the SRSF2^P95H mutation impairs the ability of the protein to read m⁵C-marked mRNA, notably reducing its binding to key leukemia-related transcripts in leukemic cells. In leukemia patients, low NSUN2 expression leads to mRNA m⁵C hypomethylation and, combined with SRSF2^P95H, predicts poor outcomes. Altogether, we highlight an unrecognized mechanistic link between epitranscriptomics and a key oncogenesis driver.