Analysis of the Ribosomal Protein S19 Interactome

Stefania Orrù,Anna Aspesi,Marta Armiraglio,Marianna Caterino,Fabrizio Loreni,Margherita Ruoppolo,Claudio Santoro,Irma Dianzani

doi:10.1074/mcp.m600156-mcp200

Abstract

Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit. Its mutations are responsible for Diamond Blackfan anemia, a congenital disease characterized by defective erythroid progenitor maturation. Dysregulation of RPS19 has therefore been implicated in this defective erythropoiesis, although the link between them is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown functions not directly connected with the structural role of RPS19 in the ribosome. A role in rRNA processing has been surmised for the yeast ortholog, whereas the extracellular RPS19 dimer has a monocyte chemotactic activity. Three proteins are known to interact with RPS19: FGF2, complement component 5 receptor 1, and a nucleolar protein called RPS19-binding protein. We have used a yeast two-hybrid approach to identify a fourth protein: the serine-threonine kinase PIM1. The present study describes our use of proteomics strategies to look for proteins interacting with RPS19 to determine its functions. Proteins were isolated by affinity purification with a GST-RPS19 recombinant protein and identified using LCMS/MS analysis coupled to bioinformatics tools. We identified 159 proteins from the following Gene Ontology categories: NTPases (ATPases and GTPases; five proteins), hydrolases/helicases (19 proteins), isomerases (two proteins), kinases (three proteins), splicing factors (five proteins), structural constituents of ribosome (29 proteins), transcription factors (11 proteins), transferases (five proteins), transporters (nine proteins), DNA/RNA-binding protein species (53 proteins), other (one dehydrogenase protein, one ligase protein, one peptidase protein, one receptor protein, and one translation elongation factor), and 13 proteins of still unknown function. Proteomics results were validated by affinity purification and Western blotting. These interactions were further confirmed by co-immunoprecipitation using a monoclonal RPS19 antibody. Many interactors are nucleolar proteins and thus are expected to take part in the RPS19 interactome; however, some proteins suggest additional functional roles for RPS19.

Highlights

Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit
To check the efficiency of the pulldown experiments, aliquots of the proteins eluted from the GST or the GST-RPS19 resins were analyzed by Western blotting using an anti-PIM1 antibody
In the same study we revealed PIM1 (15), which was not detected in the proteomics analysis despite its presence in the eluate from GST-RPS19 resin (Fig. 4) and in the immunoprecipitate obtained with the anti-RPS19 monoclonal antibody (Fig. 5)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Whole Cell Extract—Human erythroleukemia K562 cells (ATCC number CCL-243) were cultured in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37 °C with 5% CO2. Unbound proteins were incubated with the same quantity and volume of GST-RPS19 resin overnight at 4 °C on a rocker. The supernatant was incubated with an anti-RPS19 monoclonal antibody (hybridoma supernatant) and with protein G-agarose on a rocker at 4 °C for 16 h. Immunocomplexes were pelleted by centrifugation, extensively washed with Washing Buffer (TM 0.1 ϩ 0.5% Triton X-100), resuspended in SDS-PAGE Sample Buffer (750 mM Tris-HCl, pH 8.8, 5% SDS, 40% glycerol, 10% ␤-mercaptoethanol), and subjected to Western blot analysis using antibodies specific for PIM1, IGF2BP1, MCM6, DDX5, STAU1, DKC1, and NCL. SDS-PAGE, In-gel Digestion, Peptide Mapping, and Mass Spectrometry—The six pellets obtained by affinity purification were resuspended in SDS-PAGE sample buffer and pooled for one-dimensional electrophoresis. In Silico Analysis—A list of primary (direct) and secondary (indirect) protein-protein interactions of RPS19 was created using the webavailable Human Protein Reference Database (www.hprd.org). Ortholog Saccharomyces cerevisiae gene names were determined using the web-available database Ensembl (www.ensembl.org)

RESULTS

Yeast gene

MARS Peptidase activity

Dehydropyrimidine dehydrogenase

DISCUSSION