title: Study of the effects of proteasome inhibitors on ribosomal protein S19 (RPS19) mutants, identified in patients with Diamond-Blackfan anemia.
abstract: BACKGROUND
  Mutations in the ribosomal protein S19 gene (RPS19) have been found in 25% of patients with Diamond-Blackfan anemia, a rare syndrome of congenital bone marrow failure characterized by erythroblastopenia and various malformations. Mechanistic understanding of the role of RPS19 in normal erythropoiesis and in the Diamond-Blackfan anemia defect is still poor. However, defective ribosome biogenesis and, in particular, impaired 18S ribosomal RNA maturation have been documented in association with various identified RPS19 mutations. Recently, new genes, all encoding ribosomal proteins, have been found to be mutated in Diamond-Blackfan anemia, adding further support to the concept that ribosome biogenesis plays an important role in regulating erythropoiesis. We previously showed variability in the levels of expression and subcellular localization of a subset of RPS19 mutant proteins.
  
  
  METHODS
  To define the mechanistic basis for this variability better, we studied a large number of mutant proteins and characterized both RPS19 expression level using a specific antibody against RPS19 and RPS19 subcellular localization after transfection of Cos-7 cells with various green fluorescent protein-RPS19 mutants. To investigate the role of the proteasome in RPS19 degradation, we examined the effect of various proteasome inhibitors, namely lactacystin, MG132, and bortezomib on RPS19 expression and subcellular localization
  
  
  RESULTS
  We found two distinct classes of RPS19 protein defects in Diamond-Blackfan anemia based on the stability of the mutant proteins: (i) slightly decreased to normal levels of expression and normal nucleolar localization and (ii) markedly deficient expression and failure to localize to the nucleolus. All the proteasome inhibitors tested were able to restore the expression levels and normal subcellular localization of several unstable mutant proteins.
  
  
  CONCLUSIONS
  Our findings demonstrate an important role for the proteasomal degradation pathway in regulating the expression levels and nucleolar localization of certain mutant RPS19 proteins in Diamond-Blackfan anemia.
authors.affiliation: Service, d'Hématologie biologique, Hôpital, Robert Debré, 48 boulevard Sérurier, 75019 Paris, France.
authors.forename: Aurore
authors.initials: A
authors.lastname: Crétien
authors.forename: Corinne
authors.initials: C
authors.lastname: Hurtaud
authors.forename: Hélène
authors.initials: H
authors.lastname: Moniz
authors.forename: Alexis
authors.initials: A
authors.lastname: Proust
authors.forename: Isabelle
authors.initials: I
authors.lastname: Marie
authors.forename: Orianne
authors.initials: O
authors.lastname: Wagner-Ballon
authors.forename: Valérie
authors.initials: V
authors.lastname: Choesmel
authors.forename: Pierre-Emmanuel
authors.initials: PE
authors.lastname: Gleizes
authors.forename: Thierry
authors.initials: T
authors.lastname: Leblanc
authors.forename: Jean
authors.initials: J
authors.lastname: Delaunay
authors.forename: Gil
authors.initials: G
authors.lastname: Tchernia
authors.forename: Narla
authors.initials: N
authors.lastname: Mohandas
authors.forename: Lydie
authors.initials: L
authors.lastname: Da Costa