TUC-seq and BS-seq analyses of mRNA from wildtype and Nsun2-mutant mouse embryonic stem cells upon differentiation
Nsun2 catalyzes 5-methylcytosine (m5C) formation in several types of RNA, including tRNAs, mRNAs, and various non-coding RNAs. Nsun2-mediated m5C in tRNAs is thought to reduce their susceptibility to nucleolytic cleavage. Conversely, increased tRNA fragmentation and concomitant inhibition of protein synthesis may contribute to neurodevelopmental defects observed in mice and humans with Nsun2 mutations. In mRNA, m5C was shown to influence transcript stability, and both stabilizing and destabilizing effects have been documented. Here, we used the minimally invasive TUC-seq method to characterize the landscape of mRNA turnover dynamics during embryonic stem cell differentiation in wildtype and Nsun2-mutant cells. The data demonstrate that mRNA synthesis and stability undergo extensive changes during cellular differentiation. Remarkably, a large proportion of these changes do not result in altered mRNA abundance suggesting the existence of a buffering mechanism that is capable of simultaneously adjusting RNA synthesis and decay rates. We further show that loss of Nsun2 affects the dynamics of mRNA turnover but not the steady-state levels. Importantly, our data indicate that m5C in mRNA plays a limited or negligible role in regulating mRNA turnover changes resulting from Nsun2 ablation. 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.