Uzun, U.; Lund, A. H.

Ribosome heterogeneity arising from variable rRNA 2'O methylation (2'O Me) has been proposed as a potential mechanism for translational specialization, but whether such heterogeneity contributes to compartment specific translation remains unknown. Here, we systematically compare the 2'O Me landscapes of cytosolic and endoplasmic reticulum (ER)-associated ribosomes across three human cell types: HEK293 cells, H9-derived neural progenitor cells (NPCs), and neurons differentiated from these NPCs. Using detergent-based fractionation combined with RiboMeth-seq, we generate site-resolved rRNA methylation profiles for each compartment. Within each cell type, cytosolic and ER-associated ribosomes display highly similar 2'O Me patterns, with only modest compartment-specific differences observed at 18S:462 in NPCs and 28S:2043 in neurons. Across all samples, differences in 2'O Me patterns are more pronounced between cell types than between compartments. Together, these findings indicate that 2'O Me does not establish a broad ER-specific methylation signature and is unlikely to be a major determinant of ribosome localization or function at the ER.