Probing enzyme-dependent pseudouridylation using direct RNA sequencing to assess neuronal epitranscriptome plasticity.

Chemical modifications in mRNAs such as pseudouridine (psi) can regulate gene expression, although our understanding of the functional impact of individual psi modifications, especially in neuronal cells, is limited. We apply nanopore direct RNA sequencing to investigate psi dynamics under cellular perturbations in SH-SY5Y neuroblastoma cells. We identified psi sites and assigned them to psi synthase enzymes using siRNA-based knockdown, finding that TRUB1 knockdown slightly upregulates psi occupancy in PUS7 sites. Next, we studied psi occupancy changes upon differentiation and lead exposure of SH-SY5Y cells. A simple steady-state enzyme-substrate model reveals a strong correlation between psi synthase and mRNA substrate levels and psi modification frequencies. Finally, comparing psi sites across all three cellular states reveals stable and plastic modifications that are either condition-dependent or condition-independent. Our psi occupancy analysis across cell states, integrated with knockdown validation of modification sites, allows robust investigations into the dynamics and plasticity of RNA modifications.