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In mRNA, m5C was reported to influence transcript stability. However, it is unclear if it has stabilizing or destabilizing effects. To address the role of Nsun2 in mRNA stability, we characterized the landscape of mRNA turnover dynamics during embryonic stem cell (ESC) differentiation in wild-type and Nsun2-mutant cells. By using an RNA labeling approach combined with thiouridine-to-cytidine-sequencing (TUC-seq), we demonstrate that mRNA synthesis and stability undergo extensive changes during normal cellular differentiation. Remarkably, a large proportion of these changes did not result in altered mRNA abundance, providing evidence for robust transcript buffering during ESC differentiation. Importantly, also the loss of Nsun2 affected mRNA turnover dynamics but not the steady-state levels of transcripts. Furthermore, our data indicate that the effect of Nsun2 on mRNA turnover was not mediated by m5C deposition in mRNA, which is corroborated by catalysis-independent effects of Nsun2 on translation in early ESC differentiation. In conclusion, this study demonstrates that differentiation as well as loss of Nsun2 can induce changes in mRNA turnover dynamics that are independent of mRNA methylation but consistent with a buffering mechanism to maintain constant RNA levels.<\/jats:p>","DOI":"10.1093\/nar\/gkaf995","type":"journal-article","created":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T12:12:41Z","timestamp":1759925561000},"source":"Crossref","is-referenced-by-count":3,"title":["mRNA turnover dynamics are affected by cell differentiation and loss of the cytosine methyltransferase Nsun2"],"prefix":"10.1093","volume":"53","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0388-2304","authenticated-orcid":false,"given":"Isabel","family":"Delazer","sequence":"first","affiliation":[{"name":"Institute of Molecular Biology, Biocenter, Medical University of Innsbruck , 6020 Innsbruck 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