Stable Accumulation of Human Ribosomal Proteins in Rabbit Reticulocyte Expression System

Mutations in specific small subunit human ribosomal proteins (RPSs) have emerged as key contributors to severe, tissue-specific pathologies, indicating roles that extend beyond traditional protein translation within the ribosome. Notably, RPSA is associated with pancreatic cell migration and congenital asplenia, while RPS3, RPS4X, and RPS7 are linked to Diamond-Blackfan anemia (DBA). While strides have been made in understanding the nucleolar assembly of ribosomal proteins (RPs), the cytoplasmic events preceding RP translocation into the nucleus remain incompletely elucidated. This study investigated the expression and posttranslational modifications of human RPSA, RPS2, RPS3, RPS4X, RPS6, and RPS7 using the Rabbit Reticulocyte Lysate (RRL) cell-free system. The distinct advantage of RRL lies in its naturally enucleated state, allowing for the exploration of cytosolic modifications crucial for maintaining RP stability. We conducted coupled transcription/translation experiments, with comprehensive Western blot analyses and immunoprecipitations through a combination of anti-FLAG antibodies and magnetic beads, to delve into the nuances of RP expression and cytosolic posttranslational modifications. Our results demonstrated the robust and stable expression of small RPs within the RRL system, with disparate levels of abundance and stability among different RPs despite being expressed from the same promoter and under identical conditions. Intriguingly, RPSA, RPS3, and RPS6 exhibited a molecular weight of approximately 8-10 kDa higher than predicted, suggesting post-translational modification with a peptide. In parallel investigations, we detected the presence of enzymes for O-GlcNAc modification within the RRL system. Treatment with TMG, an O-GlcNAcase inhibitor, resulted in increased overall O-GlcNAcylation levels. These compelling results suggest that the RRL system is an invaluable tool for probing the intricate roles of posttranslational modifications in shaping the biochemistry and activity of RPs within the cytoplasm, distinct from the ribosomal complex. The significance of this research extends to its potential applicability in the context of ribosomopathies, with a particular emphasis on DBA, a condition characterized by the absence of red blood cells and their precursors. By scrutinizing RP biochemistry in the RRL system, we propose a relevant and accessible model to deepen our understanding of ribosomopathies. This exploration into the intricate landscape of posttranslational modifications enhances our knowledge of RP functions implicated in human diseases and paves the way for future therapeutic interventions. Financial support for this project was provided by The First-Year Innovation and Research Experience program at the University of Maryland.