Impact of O-GlcNAc Posttranslational Modification on Human Ribosomal Protein S3A During Stress

Abstract

Human Ribosomal Protein 3A (RPS3A) is part of the 40S ribosomal subunit and is responsible for cell signaling and processing of small nuclear RNA. Many proteins undergo post-translational modifications (PTMs), which impact protein interaction, regulation, and activity. RPS3A exhibits naturally occurring PTMs, including glycosylation, acetylation, and methylation. O-linked β-N-acetylglucosamine (O-GlcNAc) is a type of glycosylation that occurs on nuclear, cytoplasmic, and mitochondrial proteins. Under stress, RPS3A undergoes O-GlcNAc modification at Ser59 and Ser154. Using site-directed mutagenesis, we replaced the serines with alanines. We explored three mutants: Mutant 1 (M1), corresponding to S59A; Mutant 2 (M2), corresponding to S154A; and Double Mutant (DM), corresponding to S59A and S154A. When a human cell line is transfected with either RPS3A single mutant (M1 or M2), protein levels drop significantly compared to wild type (WT). When RPS3A DM is transfected, its protein levels are higher than those of WT. MG132 is a proteasome inhibitor that induces proteotoxic stress, preventing protein degradation. RPS3A responds to MG132-induced stress via O-GlcNAc-modification. When HEK293 cells are treated with MG132, O-GlcNAc levels and ubiquitinated proteins associated with RPS3A increase. Under proteotoxic stress, RPS3A levels show variable expression, suggesting a decrease over time; however, densitometry must be performed with more samples to quantify these changes. When the mutants are expressed in a Rabbit Reticulocyte Lysate system, M1 and M2 show no expression, whereas DM shows increased expression compared to WT, recapitulating results obtained with cells in culture, suggesting that the effects on expression are not transfection-dependent. Our results indicate that O-GlcNAc-targeted amino acids in RPS3A contribute to the protein’s stability in vivo.