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

Abstract

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.

mRNA psi profiling using nanopore DRS reveals cell type-specific pseudouridylation

“…NIP7 (Nucleolar pre-rRNA processing protein) is involved in RNA binding activity for ribosomal biogenesis. We also observed in a previous study that this position on NIP7 is relatively static in SH-SY5Y cells across multiple treatments(25). Inversely, the relative positional occupancy of the psi on SLC2A1 (chr1:42926727) ranges from 32% U-to-C error (Jurkat) to 79% (HepG2).…”

“…While SLC2A1 is present in both liver and T cells, its primary role in the liver is related to supporting glucose metabolism, whereas in T cells, SLC2A1 is crucial for immune function and response. This psi site has also been found to have stable occupancy across different treatments for SH-SY5Y cells(25).…”

“…Of these 70 sites, we applied additional filtering criteria, including a minimum of 30% U-to-C error in each cell line, to compile a final list of “housekeeping” psi sites ( Supplementary Table S3 ), used here to denote psi sites that are abundant across all cell types. Cross-validation of our 20 detected “housekeeping” sites with other methods (i.e., siRNA knockdown of a TRUB1 or PUS7 (24), CMC-mediated Illumina sequencing methods(6, 8), and RNA bisulfite labeling methods(5–7, 10, 28) produced a list of 17 orthogonally validated and conserved psi sites ( Figure 2a ). Of these, we mapped 11 sites to the CDS and 6 to the 3’ UTR ( Supplementary Figure S1a ).…”

“…Nanopore DRS raw fast5s for native HeLa replicates were sourced from NIH NCBI SRA BioProject accession PRJNA777450 (13). Nanopore DRS raw fast5s for native SH-SY5Y replicates were sourced from NIH NCBI SRA BioProject accession PRJNA1092333 (25).…”

“…Mod-p ID is a semi-quantitative technique as the frequency of the U-to-C basecalling error is sequence-dependent, and therefore, the occupancy of one site cannot be compared to that of a different site because of the U-to-C error. However, relative psi occupancies for the same site can be compared across cell lines or conditions (25). Furthermore, DRS has the potential to detect multiple modifications on a single transcript, providing a way to study multiple modifications on the same transcript (26).…”