Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts

Abstract

Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (eg, RPS19) or large (eg, RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site-mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA. IntroductionDiamond-Blackfan anemia (DBA) presents as normochromic, macrocytic anemia with reduced erythroid precursors in the BM. 1 Approximately half of DBA patients have skeletal abnormalities such as thumb malformations and growth retardation. 2 DBA is mostly diagnosed in infants less than 1 year of age, but in recent years, nonclassic cases of DBA are being diagnosed in adult patients. 1 DBA is associated with mutations in genes encoding ribosomal proteins in 55% of patients. 3 The most prominently mutated gene (in 25% of patients) is RPS19, 4 but mutations in RPS7, RPS10, RPS17, RPS24, and RPS26 in the small ribosomal subunit and in RPL5, RPL11, and RPL35A in the large ribosomal subunit have also been found. 3 The mutations cause haploinsufficiency of ribosomal proteins and lead to loss of ribosome function; this reduces general translation, as observed in lymphocytes derived from DBA patients. 5 Knockdown of RPS19 in hematopoietic progenitors either from human BM or cord blood decreases the colony-forming capacity of erythroid progenitors, whereas it affects the colony-forming capacity of myeloid progenitors to a far lesser extent. 6 Knockdown of Rps19 in mouse fetal liver-derived erythroblasts impairs their proliferation, but the differentiation of cells that survive the knockdown is not affected. 7 Because ribosome synthesis consumes up to 25% of a cell's energy, a disbalance in the synthesis of ribosomal proteins activates p53 and inhibits cell proliferation. 8 Free Rpl11 and Rpl5 bind and inhibit Mdm2, which reduces p53 ubiquitination and leads to its stabilization. Erythroid cells may ...

Loss of Tumor Suppressor RPL5/RPL11 Does Not Induce Cell Cycle Arrest but Impedes Proliferation Due to Reduced Ribosome Content and Translation Capacity

“…erythropoiesis associated with translational repression of mRNAs critical for this process, including Bag1 and Csde1 (65). Moreover, mouse embryos heterozygous for RPS6, which die in utero at embryonic day 5.5 (E5.5), can be rescued in a p53-deficient background but only to E12.5, when they succumb to impaired liver erythropoiesis (66).…”

Ribonuclease inhibitor 1 regulates erythropoiesis by controlling GATA1 translation

“…Surprisingly, the majority of clinical symptoms are related to erythropoiesis. In support of these observations, ribosomal deficiencies in DBA impair translation of transcripts essential for erythroid differentiation (5,6). How RPs regulate specific gene expression and how mutations in RPs lead to tissue-specific phenotypes are areas of active investigation.…”

Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA

“…In humans, reduced expression or partial loss of function in a number of r-proteins are associated with a group of congenital disorders termed ribosomopathies, characterized by impaired proliferation and increased cell death in hematopoietic progenitors and certain other cell lineages (Ellis and Gleizes 2011;Raiser et al 2014). On a subcellular level, defects in ribosome biogenesis lead to reduced protein synthesis capacity and misregulated translational controls (Horos et al 2012;Teng et al 2013;Ludwig et al 2014). In addition, perturbations in ribosome biogenesis can activate the tumor suppressor p53, triggering lineage-specific cell cycle arrest or apoptosis (for review, see Bursac et al 2014).…”