Stable accumulation of human ribosomal proteins SA, S2, S3, S4X, S6, S7 in rabbit reticulocyte cell-free expression system

Abstract

Mutations in human ribosomal proteins (RPs) contribute to severe, tissue-specific pathologies, indicating roles that extend beyond protein translation. RPSA is associated with pancreatic cell migration and congenital asplenia, while RPS3, RPS4X, and RPS7 are linked to Diamond-Blackfan anemia (DBA). While we understand the nucleolar assembly of RPs, the cytoplasmic events preceding RP translocation into the nucleus remain unclear. This work investigated the expression and posttranslational modifications (PTMs) of human RPSA, RPS2, RPS3, RPS4X, RPS6, and RPS7 using Rabbit Reticulocyte Lysate (RRL), an enucleated cell-free system that allows the study of cytosolic modifications crucial for RP stability. We conducted coupled transcription/translation experiments with Western blot analyses and immunoprecipitations with anti-FLAG antibodies and magnetic beads to analyze RP expression and cytosolic PTMs. Our results demonstrated the robust and stable expression of RPs, with distinct abundance and stability among different proteins despite expression from the same promoter under identical conditions. RPSA, RPS3, and RPS6 exhibited a molecular weight of approximately 8-10 kDa higher than predicted, suggesting PTM with a peptide. We also detected the presence of enzymes for O-GlcNAc modification within the RRL system. Treatment with TMG, an O-GlcNAcase inhibitor, resulted in increased O-GlcNAcylation levels. We propose that the RRL system is an invaluable tool for probing the roles of PTMs in the extraribosomal activity of RPs within the cytoplasm, representing a relevant and accessible model to deepen our understanding of ribosomopathies and their future therapeutic interventions.