Temporal and spatial profiling of ALKBH5 activity through NAIL-MS and compartmentalized RNA Isolation

RNA modifications, especially m ⁶ A in human mRNA, are believed to be dynamically regulated through RNA writers and erasers. The key eraser of m ⁶ A is ALKBH5 with its function well proven in vitro, while in vivo evidence is lacking. Here, we set out to exploit nucleic acid isotope labelling coupled mass spectrometry (NAIL-MS) in a pulse chase set-up to study the in vivo function of ALKBH5 on human RNAs. For this we purified poly(A) from whole cell total RNA and found that, steady-state m ⁶ A levels and turnover dynamics were nearly identical between WT and ALKBH5 KO, despite clear evidence of robust RNA turnover within an 8-hour labeling period. To assess whether ALKBH5 might act in a compartment-specific manner, we employed an advanced subcellular fractionation strategy, allowing for the isolation of chromatin-associated, nucleoplasmic, and cytoplasmic RNA. These analyses confirmed that m ⁶ A accumulates during transcript maturation, with levels peaking in nuclear fractions and decreasing following export to the cytoplasm, supporting the thesis m ⁶ A is a dynamic modification. Notably, however, spatial and temporal profiles of m ⁶ A distribution and decay were unaffected by ALKBH5 KO. Even in chromatin-associated and nucleolar mRNA, where co-transcriptional modification and potential demethylation would be most plausible, m ⁶ A dynamics remained indistinguishable between WT and KO cells in the NAIL-MS context. We thus conclude that ALKBH5 has no major role in mRNA m ⁶ A demethylation in HEK 293T cells grown under optimal conditions.