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1.
bioRxiv ; 2023 Feb 28.
Article in English | MEDLINE | ID: mdl-36909531

ABSTRACT

The ability of ribosomes to translate the genetic code into protein requires a finely tuned ion and solvent ecosystem. However, the lack of high-resolution structures has precluded accurate positioning of all the functional elements of the ribosome and limited our understanding of the specific role of ribosomal RNA chemical modifications in modulating ribosome function in health and disease. Here, using a new sample preparation methodology based on functionalised pristine graphene-coated grids, we solve the cryo-EM structure of the human large ribosomal subunit to a resolution of 1.67 Å. The accurate assignment of water molecules, magnesium and potassium ions in our model highlights the fundamental biological role of ribosomal RNA methylation in harnessing unconventional carbon-oxygen hydrogen bonds to establish chemical interactions with the environment and fine-tune the functional interplay with tRNA. In addition, the structures of three translational inhibitors bound to the human large ribosomal subunit at better than 2 Å resolution provide mechanistic insights into how three key druggable pockets of the ribosome are targeted and illustrate the potential of this methodology to accelerate high-throughput structure-based design of anti-cancer therapeutics.

2.
Nat Commun ; 13(1): 1562, 2022 03 23.
Article in English | MEDLINE | ID: mdl-35322020

ABSTRACT

Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1-both mutated in the leukemia predisposition disorder Shwachman-Diamond syndrome - license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. We find that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. These data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukemia predisposition disorder.


Subject(s)
Leukemia , Proteins , Animals , Leukemia/metabolism , Mammals/metabolism , Mice , Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/genetics , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Shwachman-Diamond Syndrome
3.
Blood ; 134(3): 277-290, 2019 07 18.
Article in English | MEDLINE | ID: mdl-31151987

ABSTRACT

Shwachman-Diamond syndrome (SDS) is a recessive disorder typified by bone marrow failure and predisposition to hematological malignancies. SDS is predominantly caused by deficiency of the allosteric regulator Shwachman-Bodian-Diamond syndrome that cooperates with elongation factor-like GTPase 1 (EFL1) to catalyze release of the ribosome antiassociation factor eIF6 and activate translation. Here, we report biallelic mutations in EFL1 in 3 unrelated individuals with clinical features of SDS. Cellular defects in these individuals include impaired ribosomal subunit joining and attenuated global protein translation as a consequence of defective eIF6 eviction. In mice, Efl1 deficiency recapitulates key aspects of the SDS phenotype. By identifying biallelic EFL1 mutations in SDS, we define this leukemia predisposition disorder as a ribosomopathy that is caused by corruption of a fundamental, conserved mechanism, which licenses entry of the large ribosomal subunit into translation.


Subject(s)
Mutation , Peptide Elongation Factors/genetics , Peptide Initiation Factors/biosynthesis , Ribonucleoprotein, U5 Small Nuclear/genetics , Shwachman-Diamond Syndrome/genetics , Shwachman-Diamond Syndrome/metabolism , Adolescent , Animals , Cells, Cultured , DNA Mutational Analysis , Disease Models, Animal , Disease Susceptibility , Female , Genome-Wide Association Study , Humans , Infant , Male , Mice , Mice, Transgenic , Models, Molecular , Pedigree , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/metabolism , Phenotype , Protein Conformation , Ribonucleoprotein, U5 Small Nuclear/chemistry , Ribonucleoprotein, U5 Small Nuclear/metabolism , Shwachman-Diamond Syndrome/diagnosis , Structure-Activity Relationship , Whole Genome Sequencing
4.
Mol Ther ; 25(8): 1805-1814, 2017 08 02.
Article in English | MEDLINE | ID: mdl-28434866

ABSTRACT

Diamond-Blackfan anemia is a congenital erythroid hypoplasia and is associated with physical malformations and a predisposition to cancer. Twenty-five percent of patients with Diamond-Blackfan anemia have mutations in a gene encoding ribosomal protein S19 (RPS19). Through overexpression of RPS19 using a lentiviral vector with the spleen focus-forming virus promoter, we demonstrated that the Diamond-Blackfan anemia phenotype can be successfully treated in Rps19-deficient mice. In our present study, we assessed the efficacy of a clinically relevant promoter, the human elongation factor 1α short promoter, with or without the locus control region of the ß-globin gene for treatment of RPS19-deficient Diamond-Blackfan anemia. The findings demonstrate that these vectors rescue the proliferation defect and improve erythroid development of transduced RPS19-deficient bone marrow cells. Remarkably, bone marrow failure and severe anemia in Rps19-deficient mice was cured with enforced expression of RPS19 driven by the elongation factor 1α short promoter. We also demonstrate that RPS19-deficient bone marrow cells can be transduced and these cells have the capacity to repopulate bone marrow in long-term reconstituted mice. Our results collectively demonstrate the feasibility to cure RPS19-deficient Diamond-Blackfan anemia using lentiviral vectors with cellular promoters that possess a reduced risk of insertional mutagenesis.


Subject(s)
Anemia, Diamond-Blackfan/genetics , Bone Marrow/metabolism , Bone Marrow/pathology , Genetic Vectors/genetics , Lentivirus/genetics , Promoter Regions, Genetic , Anemia, Diamond-Blackfan/diagnosis , Anemia, Diamond-Blackfan/therapy , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Bone Marrow Transplantation , Cell Differentiation/genetics , Cell Proliferation , Disease Models, Animal , Gene Expression , Gene Order , Genetic Therapy , Graft Survival/genetics , Hematopoiesis/genetics , Humans , Mice , Phenotype , RNA Interference , RNA, Small Interfering/genetics , Ribosomal Proteins/genetics , Transduction, Genetic , Transgenes , Virus Integration
5.
Br J Haematol ; 171(4): 517-29, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26305041

ABSTRACT

Diamond-Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19-deficient cells- in a disease-specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease-specific treatments of DBA.


Subject(s)
Anemia, Diamond-Blackfan/drug therapy , Erythropoiesis/drug effects , Prednisolone/therapeutic use , Ribosomal Proteins/deficiency , Tumor Suppressor Protein p53/physiology , Adolescent , Anemia, Diamond-Blackfan/blood , Animals , Apoptosis/drug effects , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Cyclin-Dependent Kinase Inhibitor p21/genetics , Dexamethasone/pharmacology , Disease Models, Animal , Drug Evaluation, Preclinical , Erythroid Precursor Cells/drug effects , Humans , Male , Mice , Mice, Inbred C57BL , Prednisolone/pharmacology , Radiation Chimera , Ribosomal Proteins/genetics , Transcription Factors/biosynthesis , Transcription Factors/genetics , Up-Regulation/drug effects , bcl-2-Associated X Protein/biosynthesis , bcl-2-Associated X Protein/genetics
6.
Cell Rep ; 9(4): 1246-55, 2014 Nov 20.
Article in English | MEDLINE | ID: mdl-25456127

ABSTRACT

Studies of developmental pathways of hematopoietic stem cells (HSCs) have defined lineage relationships throughout the blood system. This is relevant to acute myeloid leukemia (AML), where aggressiveness and therapeutic responsiveness can be influenced by the initial stage of transformation. To address this, we generated a mouse model in which the mixed-lineage leukemia/eleven-nineteen-leukemia (MLL-ENL) transcription factor can be conditionally activated in any cell type. We show that AML can originate from multiple hematopoietic progenitor subsets with granulocytic and monocytic potential, and that the normal developmental position of leukemia-initiating cells influences leukemic development. However, disease failed to arise from HSCs. Although it maintained or upregulated the expression of target genes associated with leukemic development, MLL-ENL dysregulated the proliferative and repopulating capacity of HSCs. Therefore, the permissiveness for development of AML may be associated with a narrower window of differentiation than was previously appreciated, and hijacking the self-renewal capacity of HSCs by a potent oncogene is insufficient for leukemic development.


Subject(s)
Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Cytoprotection , Hematopoietic Stem Cells/cytology , Myeloid-Lymphoid Leukemia Protein/metabolism , Oncogene Proteins, Fusion/metabolism , Animals , Carcinogenesis/drug effects , Carcinogenesis/genetics , Carcinogenesis/pathology , Cell Differentiation/drug effects , Cytoprotection/drug effects , Disease Models, Animal , Doxycycline/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Hematopoietic Stem Cells/drug effects , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Progenitor Cells/pathology , Reproducibility of Results , Transcription, Genetic/drug effects
7.
Haematologica ; 99(12): 1792-8, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25216681

ABSTRACT

Diamond-Blackfan anemia is a congenital erythroid hypoplasia caused by functional haploinsufficiency of genes encoding ribosomal proteins. Mutations involving the ribosomal protein S19 gene are detected in 25% of patients. Enforced expression of ribosomal protein S19 improves the overall proliferative capacity, erythroid colony-forming potential and erythroid differentiation of hematopoietic progenitors from ribosomal protein S19-deficient patients in vitro and in vivo following xenotransplantation. However, studies using animal models are needed to assess the therapeutic efficacy and safety of the viral vectors. In the present study we have validated the therapeutic potential of gene therapy using mouse models of ribosomal protein S19-deficient Diamond-Blackfan anemia. Using lentiviral gene transfer we demonstrated that enforced expression of ribosomal protein S19 cures the anemia and lethal bone marrow failure in recipients transplanted with ribosomal protein S19-deficient cells. Furthermore, gene-corrected ribosomal protein S19-deficient cells showed an increased pan-hematopoietic contribution over time compared to untransduced cells without signs of vector-mediated toxicity. Our study provides a proof of principle for the development of clinical gene therapy to cure ribosomal protein 19-deficient Diamond-Blackfan anemia.


Subject(s)
Anemia, Diamond-Blackfan/prevention & control , Disease Models, Animal , Genetic Therapy , Genetic Vectors/administration & dosage , Hematopoietic Stem Cells/cytology , Hemoglobinuria, Paroxysmal/prevention & control , Ribosomal Proteins/physiology , Anemia, Aplastic , Anemia, Diamond-Blackfan/genetics , Anemia, Diamond-Blackfan/pathology , Animals , Bone Marrow Diseases , Bone Marrow Failure Disorders , Hematopoietic Stem Cells/metabolism , Hemoglobinuria, Paroxysmal/genetics , Hemoglobinuria, Paroxysmal/pathology , Humans , Mice , Mice, Knockout , Mice, Transgenic , RNA, Small Interfering/genetics , Ribosomal Proteins/antagonists & inhibitors
8.
Blood ; 120(11): 2225-8, 2012 Sep 13.
Article in English | MEDLINE | ID: mdl-22791294

ABSTRACT

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Recently, a case study reported a patient who became transfusion-independent in response to treatment with the amino acid L-leucine. Therefore, we have validated the therapeutic effect of L-leucine using our recently generated mouse model for RPS19-deficient DBA. Administration of L-leucine significantly improved the anemia in Rps19-deficient mice (19% improvement in hemoglobin concentration; 18% increase in the number of erythrocytes), increased the bone marrow cellularity, and alleviated stress hematopoiesis. Furthermore, the therapeutic response to L-leucine appeared specific for Rps19-deficient hematopoiesis and was associated with down-regulation of p53 activity. Our study supports the rationale for clinical trials of L-leucine as a therapeutic agent for DBA.


Subject(s)
Anemia, Diamond-Blackfan/diet therapy , Dietary Supplements , Disease Models, Animal , Hematinics/therapeutic use , Hematopoiesis , Leucine/therapeutic use , Up-Regulation , Anemia, Diamond-Blackfan/blood , Anemia, Diamond-Blackfan/metabolism , Anemia, Diamond-Blackfan/pathology , Animals , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Down-Regulation , Erythrocyte Count , Gene Knockdown Techniques , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Hemoglobins/analysis , Mice , Mice, Transgenic , Molecular Targeted Therapy , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering , Ribosomal Proteins/antagonists & inhibitors , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
9.
Blood ; 118(23): 6087-96, 2011 Dec 01.
Article in English | MEDLINE | ID: mdl-21989989

ABSTRACT

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.


Subject(s)
Anemia, Diamond-Blackfan/genetics , Disease Models, Animal , Hemoglobinuria, Paroxysmal/genetics , Mice, Transgenic , Ribosomal Proteins/genetics , Anemia, Aplastic , Anemia, Diamond-Blackfan/pathology , Anemia, Diamond-Blackfan/physiopathology , Anemia, Macrocytic/genetics , Anemia, Macrocytic/pathology , Anemia, Macrocytic/physiopathology , Animals , Apoptosis/physiology , Bone Marrow Diseases , Bone Marrow Failure Disorders , Bone Marrow Transplantation , Cell Division/physiology , Cells, Cultured , Gene Expression/physiology , Hematopoietic Stem Cells/pathology , Hematopoietic Stem Cells/physiology , Hemoglobinuria, Paroxysmal/pathology , Hemoglobinuria, Paroxysmal/physiopathology , Leukopenia/genetics , Leukopenia/pathology , Leukopenia/physiopathology , Mice , Phenotype , Platelet Count , RNA, Small Interfering/pharmacology , Ribosomal Proteins/deficiency , Tumor Suppressor Protein p53/genetics
10.
Blood ; 115(23): 4689-98, 2010 Jun 10.
Article in English | MEDLINE | ID: mdl-20371744

ABSTRACT

Numerous publications have described the importance of bone morphogenetic protein (BMP) signaling in the specification of hematopoietic tissue in developing embryos. Here we investigate the full role of canonical BMP signaling in both adult and fetal liver hematopoiesis using conditional knockout strategies because conventional disruption of components of the BMP signaling pathway result in early death of the embryo. By targeting both Smad1 and Smad5, we have generated a double-knockout mouse with complete disruption of canonical BMP signaling. Interestingly, concurrent deletion of Smad1 and Smad5 results in death because of extrahematopoietic pathologic changes in the colon. However, Smad1/Smad5-deficient bone marrow cells can compete normally with wild-type cells and display unaffected self-renewal and differentiation capacity when transplanted into lethally irradiated recipients. Moreover, although BMP receptor expression is increased in fetal liver, fetal liver cells deficient in both Smad1 and Smad5 remain competent to long-term reconstitute lethally irradiated recipients in a multilineage manner. In conclusion, canonical BMP signaling is not required to maintain either adult or fetal liver hematopoiesis, despite its crucial role in the initial patterning of hematopoiesis in early embryonic development.


Subject(s)
Bone Morphogenetic Proteins/metabolism , Fetus/embryology , Hematopoiesis, Extramedullary/physiology , Hematopoietic Stem Cells/metabolism , Liver/embryology , Signal Transduction/physiology , Animals , Bone Morphogenetic Protein Receptors/biosynthesis , Bone Morphogenetic Protein Receptors/genetics , Bone Morphogenetic Proteins/genetics , Cell Differentiation/physiology , Colon/embryology , Colon/metabolism , Embryo Loss/genetics , Embryo Loss/metabolism , Gene Expression Regulation, Developmental/physiology , Hematopoietic Stem Cell Transplantation , Liver/metabolism , Mice , Mice, Knockout , Smad1 Protein/genetics , Smad1 Protein/metabolism , Smad5 Protein/genetics , Smad5 Protein/metabolism , Transplantation, Homologous
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