Linking Defective Ribosome Maturation to Shwachman-Diamond Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-36-SCI-36
Author(s):  
Alan John Warren
Keyword(s):  
Ribosome Biogenesis ◽  
Conflicts Of Interest ◽  
Ribosomal Subunit ◽  
Protein Translation ◽  
Ribosome Assembly ◽  
Genetic Dissection ◽  
Gtp Hydrolysis ◽  
Shwachman Diamond Syndrome ◽  
Novel Therapeutic Strategies ◽  
Syndrome Protein

Abstract Ribosomes are RNA-protein machines that translate the genetic information encoded by the mRNA template in all living cells. Recent high-resolution structures of the ribosome have revolutionized our understanding of protein translation. However, the mechanisms of ribosome assembly and the surveillance mechanisms that monitor this process and couple it to growth are poorly understood. Causative mutations and deletions of genes involved in ribosome biogenesis define an emerging group of disorders known as the ribosomopathies. Recent work from my laboratory strongly supports the hypothesis that Shwachman-Diamond syndrome (SDS) is a ribosomopathy caused by defective maturation of the large ribosomal subunit. Elucidation of the specific function of the SBDS protein that is deficient in SDS is revealing unexpected new insights that extend our understanding of the mechanisms underlying the late cytoplasmic steps of ribosome assembly and the quality control surveillance pathways that monitor 60S maturation. Genetic dissection of this pathway may inform novel therapeutic strategies for SDS. 1. Wong C.C., Traynor D., Basse N., Kay R.R., Warren A.J. Defective ribosome assembly in Shwachman-Diamond syndrome. Plenary Paper, Blood. 2011 Oct 20;118(16):4305-12. 2. Finch A.J., Hilcenko C., Basse N., Drynan L.F., Goyenechea B., Menne T.F., González Fernández Á., Simpson P., D’Santos C.S., Arends M.J., Donadieu J., Bellanné-Chantelot C., Costanzo M., Boone C., McKenzie A.N., Freund S.M., Warren A.J. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes and Development (2011) 25: 917-929. 3. Menne T.M., Goyenechea B., Sánchez-Puig N., Wong C.C., Tonkin L.M., Ancliff P., Brost R.L., Costanzo M., Boone C. and Warren A.J. The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast. Nature Genetics (2007) 39: 486-95. Disclosures: No relevant conflicts of interest to declare.

SBDS and eIF6 Modulate Ribosome Subunit Joining in Shwachman Diamond Syndrome,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3438-3438
Author(s):  
Nicholas Burwick ◽  
Scott Coats ◽  
Akiko Shimamura
Keyword(s):  
Stromal Cells ◽  
Ribosome Biogenesis ◽  
Conflicts Of Interest ◽  
Ribosomal Subunit ◽  
Marrow Stromal Cells ◽  
Vitro Assay ◽  
Amino Terminal ◽  
Shwachman Diamond Syndrome ◽  
Subunit Joining

Abstract Abstract 3438 Shwachman Diamond syndrome (SDS) is an autosomal recessive marrow failure syndrome with a predisposition to leukemia. Over 90% of SDS patients harbor biallelic mutations in the SBDS gene. SBDS has been implicated in several cellular functions including ribosome biogenesis and mitotic spindle stabilization. Deletion of SBDS orthologues in yeast results in a severe slow growth phenotype and depressed polysomes. Homozygous deletion of Sbds in murine models results in early embryonic lethality, while conditional deletion of Sbds in mouse liver demonstrates accumulation of 40S and 60S subunits and halfmer formation consistent with impaired ribosome joining. SBDS facilitates the release of eIF6, a factor that prevents ribosome joining. The dramatic phenotypic and polysome changes noted in these experimental models were not observed in cells derived from SDS patients. SDS patient cells have only a mildly reduced growth rate compared to heatlhy controls, and polysome profiles do not demonstrate depressed polysomes or halfmer formation. Since complete abrogation of SBDS expression is lethal and biallelic null mutations in SBDS have not been reported, we examined the role of SBDS and eIF6 in SDS patients and human cell models. We first investigated whether ribosome subunit homeostasis is impaired in SDS patient cells. We find that the 60S:40S ribosomal subunit ratio is consistently reduced in bone marrow stromal cells from SDS patients of different genotypes (n=4). This impairment in 60S:40S ratio is demonstrated in both SDS patient stromal cells and patient lymphoblasts. Stable lentiviral knockdown of SDS in normal marrow stromal cells recapitulates the reduction in 60S:40S ratio. SBDS and eIF6 co-sediment in polysome gradients of human SDS cells. This co-sedimentation is specific for the 60S ribosomal subunit. Since eIF6 has a role as an anti-joining factor, we next developed an in vitro assay to test for ribosome subunit joining in human cells. In this assay, we validate that over-expression of eIF6 results in reduced ribosome joining, and eIF6 knockdown promotes ribosome joining. Moreover, we find that SDS patient stromal cells and patient lymphoblasts both demonstrate impaired ribosome subunit joining, compared with healthy controls. Importantly, the addition of wild type SBDS or depletion of eIF6 improve ribosome joining in SDS patient cells. We demonstrate that the amino terminal sequences of SBDS are necessary but not sufficient for the association of SBDS with the 60S ribosomal subunit. Insertion of a patient-derived N-terminal SBDS point mutation also results in decreased association of SBDS with the 60S ribosomal subunit. These structure-function studies may help to inform genotype:phenotype correlations in SDS. The role of defective ribosome joining in promoting the SDS hematopoietic phenotype is of particular interest. Ongoing studies are interrogating the role of eIF6 modulation on the hematopoietic phenotype in SBDS- depleted cells. Insights garnered from these experiments will help inform the development of novel agents to improve the hematopoetic defect in human SDS. Disclosures: No relevant conflicts of interest to declare.

Shwachman-Diamond Syndrome and the Quality Control of Ribosome Assembly

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-42-SCI-42
Author(s):  
Alan J. Warren
Keyword(s):  
Quality Control ◽  
Ribosomal Proteins ◽  
Active Sites ◽  
Structural Integrity ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Final Maturation ◽  
Shwachman Diamond Syndrome

Abstract The synthesis of new ribosomes is a fundamental conserved process in all cells. Ribosomes are pre-assembled in the nucleus and subsequently exported to the cytoplasm where they acquire functionality through a series of final maturation steps that include formation of the catalytic center, recruitment of the last remaining ribosomal proteins and the removal of inhibitory assembly factors. Surprisingly, a number of key factors (SBDS, DNAJC21, RPL10 (uL16)) involved in late cytoplasmic maturation of the large (60S) ribosomal subunit are mutated in both inherited and sporadic forms of leukemia. In particular, biallelic mutations in the SBDS gene cause Shwachman-Diamond syndrome (SDS), a recessive bone marrow failure disorder with significant predisposition to acute myeloid leukemia. By using the latest advances in single-particle cryo-electron microscopy to elucidate the function of the SBDS protein, we have uncovered an elegant mechanism that couples final maturation of the 60S subunit to a quality control assessment of the structural integrity of the active sites of the ribosome. Further molecular dissection of this pathway may inform novel therapeutic strategies for SDS and leukemia more generally. References: 1. Weis F, Giudice E, Churcher M,et al. Mechanism of eIF6 release from the nascent 60S ribosomal subunit. Nat Struct Mol Biol, (2015) Nov;22(11):914-9. 2. Wong CC, Traynor D, Basse N, et al. Defective ribosome assembly in Shwachman-Diamond syndrome. Plenary Paper, Blood. 2011 Oct 20;118(16):4305-12. 3. Finch AJ, Hilcenko C, Basse N, et al. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes Dev (2011) 25: 917-929. 4. Menne TM, Goyenechea B, Sánchez-Puig N, et al. The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast. Nature Genetics (2007) 39: 486-95. Disclosures No relevant conflicts of interest to declare.

scholarly journals Defective ribosome assembly in Shwachman-Diamond syndrome

Blood ◽  
2011 ◽  
Vol 118 (16) ◽  
pp. 4305-4312 ◽  
Author(s):  
Chi C. Wong ◽  
David Traynor ◽  
Nicolas Basse ◽  
Robert R. Kay ◽  
Alan J. Warren
Keyword(s):  
Ribosome Biogenesis ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Ribosome Assembly ◽  
Restrictive Temperature ◽  
Poor Growth ◽  
Shwachman Diamond Syndrome ◽  
Subunit Joining

AbstractShwachman-Diamond syndrome (SDS), a recessive leukemia predisposition disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, skeletal abnormalities and poor growth, is caused by mutations in the highly conserved SBDS gene. Here, we test the hypothesis that defective ribosome biogenesis underlies the pathogenesis of SDS. We create conditional mutants in the essential SBDS ortholog of the ancient eukaryote Dictyostelium discoideum using temperature-sensitive, self-splicing inteins, showing that mutant cells fail to grow at the restrictive temperature because ribosomal subunit joining is markedly impaired. Remarkably, wild type human SBDS complements the growth and ribosome assembly defects in mutant Dictyostelium cells, but disease-associated human SBDS variants are defective. SBDS directly interacts with the GTPase elongation factor-like 1 (EFL1) on nascent 60S subunits in vivo and together they catalyze eviction of the ribosome antiassociation factor eukaryotic initiation factor 6 (eIF6), a prerequisite for the translational activation of ribosomes. Importantly, lymphoblasts from SDS patients harbor a striking defect in ribosomal subunit joining whose magnitude is inversely proportional to the level of SBDS protein. These findings in Dictyostelium and SDS patient cells provide compelling support for the hypothesis that SDS is a ribosomopathy caused by corruption of an essential cytoplasmic step in 60S subunit maturation.

scholarly journals The GTPase BipA expressed at low temperature in Escherichia coli assists ribosome assembly and has chaperone-like activity

2018 ◽  
Vol 293 (47) ◽  
pp. 18404-18419 ◽  
Author(s):  
Eunsil Choi ◽  
Jihwan Hwang
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Ribosomal Protein ◽  
Ribosome Biogenesis ◽  
Cold Shock ◽  
Ribosomal Subunit ◽  
Protein Translation ◽  
Receptor Protein ◽  
Deletion Strain ◽  
Ribosome Assembly

BPI-inducible protein A (BipA) is a conserved ribosome-associated GTPase in bacteria that is structurally similar to other GTPases associated with protein translation, including IF2, EF-Tu, and EF-G. Its binding site on the ribosome appears to overlap those of these translational GTPases. Mutations in the bipA gene cause a variety of phenotypes, including cold and antibiotics sensitivities and decreased pathogenicity, implying that BipA may participate in diverse cellular processes by regulating translation. According to recent studies, a bipA-deletion strain of Escherichia coli displays a ribosome assembly defect at low temperature, suggesting that BipA might be involved in ribosome assembly. To further investigate BipA's role in ribosome biogenesis, here, we compared and analyzed the ribosomal protein compositions of MG1655 WT and bipA-deletion strains at 20 °C. Aberrant 50S ribosomal subunits (i.e. 44S particles) accumulated in the bipA-deletion strain at 20 °C, and the ribosomal protein L6 was absent in these 44S particles. Furthermore, bipA expression was significantly stimulated at 20 °C, suggesting that it encodes a cold shock–inducible GTPase. Moreover, the transcriptional regulator cAMP receptor protein (CRP) positively promoted bipA expression only at 20 °C. Importantly, GFP and α-glucosidase refolding assays revealed that BipA has chaperone activity. Our findings indicate that BipA is a cold shock–inducible GTPase that participates in 50S ribosomal subunit assembly by incorporating the L6 ribosomal protein into the 44S particle during the assembly.

SBDS Protein Plays a Role in Ribosome Biogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 185-185
Author(s):  
Karthik A. Ganapathi ◽  
Karyn M. Austin ◽  
Maggie Malsch ◽  
Akiko Shimamura
Keyword(s):  
Ribosome Biogenesis ◽  
Actinomycin D ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Protein Translation ◽  
Exocrine Pancreatic Insufficiency ◽  
Nucleolar Localization ◽  
28S Rrna ◽  
Wild Type ◽  
Shwachman Diamond Syndrome

Abstract Shwachman-Diamond syndrome is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow failure, and leukemia predisposition. The majority of patients with Shwachman-Diamond syndrome harbor mutations in the SBDS gene. SBDS is a novel gene of unknown function and is highly conserved throughout evolution. Studies of the yeast orthologue, YLR022c/SDO1, suggest that SBDS may play a role in ribosome biogenesis. In support of this hypothesis, we have found that the SBDS protein shuttles in and out of the nucleolus. Previously we have shown that SBDS nucleolar localization is regulated in a cell cycle-dependant manner. We now find that SBDS nucleolar localization is also lost following exposure to actinomycin D, suggesting that SBDS nucleolar localization is dependent on active ribosomal RNA (rRNA) transcription. In cell survival assays, SBDS−/− patient-derived cells are sensitive to actinomycin D treatment relative to normal control cells. Introduction of the wild-type SBDS cDNA into SBDS−/− cells corrects their actinomycin D sensitivity, confirming that the observed sensitivity is SBDS-dependent. In contrast, SBDS−/− cells do not exhibit increased sensitivity to cyclohexamide, a protein translation inhibitor. Consistent with this result, SBDS protein co-localizes with ribosomal precursor subunits but not with mature polysomes upon sucrose gradient sedimentation. No differences in polysome profiles are observed between SBDS−/− cells and wild type control cells. Gel filtration studies suggest that SBDS associates into a complex with other proteins. SBDS co-immunoprecipitates with other nucleolar proteins involved in rRNA biogenesis. RNA immunoprecipitation studies reveal that SBDS also associates with the 28S rRNA but not the 18S rRNA. These findings support the hypothesis that SBDS plays a role in ribosome biogenesis

scholarly journals The Impact of the Stringent Response on TRAFAC GTPases and Prokaryotic Ribosome Assembly

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1313 ◽  
Author(s):  
Bennison ◽  
Irving ◽  
Corrigan
Keyword(s):  
Ribosomal Proteins ◽  
Cellular Response ◽  
Ribosome Biogenesis ◽  
Stringent Response ◽  
Protein Translation ◽  
Bound State ◽  
Ribosome Assembly ◽  
Rrna Transcription ◽  
Stress Sensing ◽  
The Impact

Many facets of ribosome biogenesis and function, including ribosomal RNA (rRNA) transcription, 70S assembly and protein translation, are negatively impacted upon induction of a nutrient stress-sensing signalling pathway termed the stringent response. This stress response is mediated by the alarmones guanosine tetra- and penta-phosphate ((p)ppGpp), the accumulation of which leads to a massive cellular response that slows growth and aids survival. The 70S bacterial ribosome is an intricate structure, with assembly both complex and highly modular. Presiding over the assembly process is a group of P-loop GTPases within the TRAFAC (Translation Factor Association) superclass that are crucial for correct positioning of both early and late stage ribosomal proteins (r-proteins) onto the rRNA. Often described as ‘molecular switches’, members of this GTPase superfamily readily bind and hydrolyse GTP to GDP in a cyclic manner that alters the propensity of the GTPase to carry out a function. TRAFAC GTPases are considered to act as checkpoints to ribosome assembly, involved in binding to immature sections in the GTP-bound state, preventing further r-protein association until maturation is complete. Here we review our current understanding of the impact of the stringent response and (p)ppGpp production on ribosome maturation in prokaryotic cells, focusing on the inhibition of (p)ppGpp on GTPase-mediated subunit assembly, but also touching upon the inhibition of rRNA transcription and protein translation.

Impaired Ribosome Maturation In Human Cells Depleted of Shwachman-Diamond Syndrome Protein SBDS

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2242-2242
Author(s):  
Gulay Sezgin ◽  
Abdallah Nihrane ◽  
Adrianna Henson ◽  
Max Wattenberg ◽  
Steven Ellis ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Ribosomal Subunit ◽  
A549 Cells ◽  
Autosomal Recessive Disorder ◽  
Human Cells ◽  
Yeast Cells ◽  
Cycle Phase ◽  
Shwachman Diamond Syndrome ◽  
Pancreatic Exocrine Dysfunction

Abstract Abstract 2242 Background: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, neurocognitive and skeletal abnormalities, and bone marrow failure. Mutations in SBDS have been shown to cause SDS. From experiments on its yeast ortholog (Haematologica 2010. 95:57-64), SBDS has been implicated in maturation and function of the 60S ribosomal subunit. In particular, subunit maturation in the SDS yeast model was associated with delayed export and accumulation of 60S-like particles in the nucleoplasm. Methods and Results: To clarify its role in human cells, erythroleukemia TF-1 cells were transduced with lentiviral vectors expressing short hairpin RNA (shRNA) against SBDS. Immunoblot assays confirmed approximately 60% knockdown in individual TF-1 cell clones expressing different shRNAs. The growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to cells stably transduced with shRNA against a scrambled SBDS sequence. Using Hoechst 33342/Pyronin Y staining and flow cytometry, we also found an increased percentage of knockdown cells retained at the G0/G1 cell cycle phase. To address whether near-complete knockdown of SBDS affected ribosome synthesis as it does in yeast cells, we silenced SBDS in A549 cells. Our data revealed a reduction in polysomes but in contrast to what was observed in yeast, there was no evidence of half-mer polysomes indicative of decreased 60S subunits participating in translation. The absence of half-mers is not unusual in mammalian systems, so to better analyze the effect of SBDS on 60S subunit maturation subunit localization was assessed by co-transfection with a vector expressing a fusion between human RPL29 and enhanced GFP. Preliminary studies indicated a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits, when compared with normal controls (Fig. 1). Conclusions: Disclosures: No relevant conflicts of interest to declare.

scholarly journals Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast

2017 ◽  
Vol 474 (2) ◽  
pp. 195-214 ◽  
Author(s):  
Salini Konikkat ◽  
John L. Woolford,
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Small Nucleolar Rnas ◽  
Large Ribosomal Subunit ◽  
Yeast Saccharomyces Cerevisiae ◽  
Subunit Assembly ◽  
Nucleolar Rnas

Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ∼76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae. We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly.

scholarly journals Conformational switches control early maturation of the eukaryotic small ribosomal subunit

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mirjam Hunziker ◽  
Jonas Barandun ◽  
Olga Buzovetsky ◽  
Caitlin Steckler ◽  
Henrik Molina ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Conformational Changes ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Early Maturation ◽  
External Transcribed Spacer ◽  
Assembly Factors

Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5’ external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5’ external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar pre-ribosomal assembly - the 5’ external transcribed spacer ribonucleoprotein – provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly.

Characterization of the Hematologic Phenotype of rpS6 Heterozygous Null Mice as a Model of the Erythroid Failure In Diamond-Blackfan Anemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2033-2033
Author(s):  
Sioban B. Keel ◽  
Janis L. Abkowitz
Keyword(s):  
Ribosomal Protein ◽  
Ribosome Biogenesis ◽  
Conflicts Of Interest ◽  
Macrocytic Anemia ◽  
Ribosomal Subunit ◽  
T Test ◽  
Ribosomal Protein Genes ◽  
Diamond Blackfan Anemia ◽  
Student’S T ◽  
Student’S T Test

Abstract Abstract 2033 Diamond-Blackfan Anemia (DBA) is a congenital form of pure red cell aplasia characterized by a hypoproliferative, macrocytic anemia, congenital anomalies, and a predisposition to cancer. DBA, along with a growing number of human diseases, is linked to defects in ribosome biogenesis. Mutations in at least 10 ribosomal protein genes of both the 40S and 60S ribosomal subunits have now been identified in over 50% of patients with DBA (Narla A, et al. Blood 2010; 115) resulting in ribosomal protein haploinsufficency and in turn a defect in ribosome biogenesis. It remains, however, unknown how these events culminate in erythroid marrow failure. The study of this pathophysiology has been hindered by a lack of animal models. We became aware of the Rps6-deleted mouse as a potential murine model of DBA (Volarevic S, et al. Science 2000; 288). RPS6 is another 40S ribosomal subunit protein required for ribosomal subunit assembly. Haploinsufficiency of RPS6 causes a phenotype reminiscent of DBA during embryogenesis (Panic L, et al. Mol Cell Biol 2006; 26), however, the erythropoietic phenotype of the conditionally-deleted Rps6 heterozygous mouse was unknown. The purpose of these studies is to fully characterize the erythroid phenotype of this mouse as a model of DBA. We demonstrate that deletion of one Rps6 allele in mice results in a macrocytic anemia and leukopenia (an absolute neutropenia and lymphocytopenia, Table 1). Though this finding is not typical, neutropenia has been described in DBA. Like DBA, the anemia is hypoproliferative (corrected reticulocyte counts were equivalent in rpS6 heterozygous and control mice: 3.3% ± 0.21, n= 3 vs. 3.6 ± 0.33, n=3; two-tailed Student's t-test, p= 0.08, which is an inappropriately low value given the deleted animals’ anemia). Flow cytometric analyses of bone marrow and spleen double-stained for Ter119 and transferrin receptor (CD71) demonstrate impaired early erythroid differentiation, evidenced by a relative expansion in the proerythroblast and basophilic erythroblast populations. Hematopoietic colony assays confirm this early defect. These data suggest that haploinsufficiency of rpS6 impacts both erythropoiesis and granulopoiesis, and since the mice are not thrombocytopenic, the effect appears lineage specific, rather than occurring in a common progenitor cell. Polysome profiles to confirm a defect in ribosome biogenesis are pending. Since heterozygous mice recapitulate the erythroid phenotype of DBA, we treated the mice with standard and potential DBA therapies. Specifically, mice received 2 mg/kg/day of prednisone for 12 weeks. There was no improvement in the hemoglobin or MCV in treated animals. As DBA and 5q- syndrome myelodysplastic syndrome (MDS) share an erythroid phenotype and both result from a haploinsufficiency of a ribosomal protein, we also tested whether the macrocytic anemia in rpS6 heterozygous mice responds to lenalidomide (Revlimid®, gift from Celgene Corporation, San Diego, CA). Mice received 3 mg/kg/day of lenalidomide by oral gavage for 12 weeks. The hemoglobin increased in control mice and markedly increased in rpS6 heterozygous mice after 12 weeks of therapy (13.5 ± 0.4 to 14.9 ± 0.2, p= 0.0 and 7.9 g/dL ± 0.9 to 10.3 ± 0.8, p= 0.01, respectively; mean ± SEM, Student's t-test, paired). Additionally, the MCV decreased with therapy in both groups (49.1 fL ± 1.4 to 41.1 ± 0.2, p=0.005 and 57.4 ± 1.1 to 53.77 ± 1.4, p=0.08). With the caveat that we did not monitor drug levels achieved in vivo, these data suggest that lenalidomide improves hemoglobinization and deserves further study in DBA. Disclosures: No relevant conflicts of interest to declare.

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