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ABSTRACT: Pseudouridine is the most prevalent RNA modification, and its aberrant function is implicated in various human diseases. However, the specific impact of pseudouridylation on hematopoiesis remains poorly understood. Here, we investigated the role of transfer RNA (tRNA) pseudouridylation in erythropoiesis and its association with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA) pathogenesis. By using patient-specific induced pluripotent stem cells (iPSCs) carrying a genetic pseudouridine synthase 1 (PUS1) mutation and a corresponding mutant mouse model, we demonstrated impaired erythropoiesis in MLASA-iPSCs and anemia in the MLASA mouse model. Both MLASA-iPSCs and mouse erythroblasts exhibited compromised mitochondrial function and impaired protein synthesis. Mechanistically, we revealed that PUS1 deficiency resulted in reduced mitochondrial tRNA levels because of pseudouridylation loss, leading to aberrant mitochondrial translation. Screening of mitochondrial supplements aimed at enhancing respiration or heme synthesis showed limited effect in promoting erythroid differentiation. Interestingly, the mammalian target of rapamycin (mTOR) inhibitor rapamycin facilitated erythroid differentiation in MLASA-iPSCs by suppressing mTOR signaling and protein synthesis, and consistent results were observed in the MLASA mouse model. Importantly, rapamycin treatment partially ameliorated anemia phenotypes in a patient with MLASA. Our findings provide novel insights into the crucial role of mitochondrial tRNA pseudouridylation in governing erythropoiesis and present potential therapeutic strategies for patients with anemia facing challenges related to protein translation.
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Eritropoese , Células-Tronco Pluripotentes Induzidas , Mitocôndrias , RNA de Transferência , Animais , Camundongos , Humanos , RNA de Transferência/genética , RNA de Transferência/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Pseudouridina/metabolismo , Anemia Sideroblástica/genética , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patologia , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , Hidroliases/metabolismo , Hidroliases/genética , Síndrome MELAS/genética , Síndrome MELAS/patologia , Síndrome MELAS/metabolismo , Modelos Animais de DoençasRESUMO
Molecular genetic changes in acute myeloid leukemia (AML) play crucial roles in leukemogenesis, including recurrent chromosome translocations, epigenetic/spliceosome mutations and transcription factor aberrations. Six1, a transcription factor of the Sine oculis homeobox (Six) family, has been shown to transform normal hematopoietic progenitors into leukemia in cooperation with Eya. However, the specific role and the underlying mechanism of Six1 in leukemia maintenance remain unexplored. Here, we showed increased expression of SIX1 in AML patients and murine leukemia stem cells (c-Kit+ cells, LSCs). Importantly, we also observed that a higher level of Six1 in human patients predicts a worse prognosis. Notably, knockdown of Six1 significantly prolonged the survival of MLL-AF9-induced AML mice with reduced peripheral infiltration and tumor burden. AML cells from Six1-knockdown (KD) mice displayed a significantly decreased number and function of LSC, as assessed by the immunophenotype, colony-forming ability and limiting dilution assay. Further analysis revealed the augmented apoptosis of LSC and decreased expression of glycolytic genes in Six1 KD mice. Overall, our data showed that Six1 is essential for the progression of MLL-AF9-induced AML via maintaining the pool of LSC.
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Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/patologia , Proteína de Leucina Linfoide-Mieloide/genética , Células-Tronco Neoplásicas/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Regulação para Cima , Animais , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Neoplasias Experimentais , Células-Tronco Neoplásicas/metabolismo , PrognósticoRESUMO
Pseudouridylation plays a regulatory role in various physiological and pathological processes. A prime example is the mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA), characterized by defective pseudouridylation resulting from genetic mutations in pseudouridine synthase 1 (PUS1). However, the roles and mechanisms of pseudouridylation in normal erythropoiesis and MLASA-related anemia remain elusive. We established a mouse model carrying a point mutation (R110W) in the enzymatic domain of PUS1, mimicking the common mutation in human MLASA. Pus1-mutant mice exhibited anemia at 4 weeks old. Impaired mitochondrial oxidative phosphorylation was also observed in mutant erythroblasts. Mechanistically, mutant erythroblasts showed defective pseudouridylation of targeted tRNAs, altered tRNA profiles, decreased translation efficiency of ribosomal protein genes, and reduced globin synthesis, culminating in ineffective erythropoiesis. Our study thus provided direct evidence that pseudouridylation participates in erythropoiesis in vivo. We demonstrated the critical role of pseudouridylation in regulating tRNA homeostasis, cytoplasmic translation, and erythropoiesis.
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Long non-coding RNA (lncRNA) has become an important regulator of many cellular processes, including cell proliferation. Although studies have shown that a variety of lncRNAs play an important role in the occurrence and development of hematopoietic malignancies, a more comprehensive and unbiased method to study the function of lncRNAs in leukemia cell lines is lacking. Here, we used short hairpin RNA (shRNA) library combined with high-throughput sequencing to screen lncRNAs that may affect the proliferation of leukemia cell lines, and identified lncRNA C20orf204-203 among 74 candidate lncRNAs in this study. Further experiments showed that C20orf204-203 was localized in the cytoplasm in both K562 and THP-1 cell lines. C20orf204-203 knockdown decreased the proliferation of K562 and THP-1 cell lines accompanied with the increased proportion of early apoptotic cells. We observed the increased mRNA level of BAD gene while decreased protein level of TP53 and BCL2. The expression of Caspase 3 decreased and Caspase 3-cleaved protein increased in THP-1 cell line. However, their changes were inconsistent in the two cell lines. Our experimental results showed that knockdown of lncRNA C20orf204-203 in leukemia cell lines affected cell proliferation although the mechanism of action in different cell lines may differ. Importantly, our research demonstrated the feasibility of using shRNA library combined with high-throughput sequencing to study the role of lncRNA in leukemia cell lines on a large scale.
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Leucemia , RNA Longo não Codificante , Caspase 3 , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Lentivirus/genética , Leucemia/genética , Proteínas Proto-Oncogênicas c-bcl-2 , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro , RNA Interferente Pequeno/genéticaRESUMO
SETD5 mutations were identified as the genetic causes of neurodevelopmental disorders. While the whole-body knockout of Setd5 in mice leads to embryonic lethality, the role of SETD5 in adult stem cell remains unexplored. Here, a critical role of Setd5 in hematopoietic stem cells (HSCs) is identified. Specific deletion of Setd5 in hematopoietic system significantly increased the number of immunophenotypic HSCs by promoting HSC proliferation. Setd5-deficient HSCs exhibited impaired long-term self-renewal capacity and multiple-lineage differentiation potentials under transplantation pressure. Transcriptome analysis of Setd5-deficient HSCs revealed a disruption of quiescence state of long-term HSCs, a cause of the exhaustion of functional HSCs. Mechanistically, SETD5 was shown to regulate HSC quiescence by mediating the release of promoter-proximal paused RNA polymerase II (Pol II) on E2F targets in cooperation with HCF-1 and PAF1 complex. Taken together, these findings reveal an essential role of SETD5 in regulating Pol II pausing-mediated maintenance of adult stem cells.
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Células-Tronco Hematopoéticas , RNA Polimerase II , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Homeostase , Humanos , Metiltransferases , Camundongos , Camundongos Knockout , RNA Polimerase II/genética , Fatores de TranscriçãoRESUMO
BACKGROUND: Fanconi anemia (FA) is a rare disease of bone marrow failure. FA patients are prone to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, the molecular clonal evolution of the progression from FA to MDS/AML remains elusive. METHODS: Herein, we performed a comprehensive genomic analysis using an FA patient (P1001) sample that transformed to MDS and subsequently AML, together with other three FA patient samples at the MDS stage. RESULTS: Our finding showed the existence of polyclonal pattern in these cases at MDS stage. The clonal evolution analysis of FA case (P1001) showed the mutations of UBASH3A, SF3B1, RUNX1 and ASXL1 gradually appeared at the later stage of MDS, while the IDH2 alteration become the dominant clone at the leukemia stage. Moreover, single-cell sequencing analyses further demonstrated a polyclonal pattern was present at either MDS or AML stages, whereas IDH2 mutated cell clones appeared only at the leukemia stage. CONCLUSIONS: We thus propose a clonal evolution model from FA to MDS and AML for this patient. The results of our study on the clonal evolution and mutated genes of the progression of FA to AML are conducive to understanding the progression of the disease that still perplexes us.
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AbstractObjective: To establish an acquired aplastic anemia animal model for investigating the function of T lymphocyte and the pathogenesis and treatment of aplastic anemia(AA). METHODS: To establish the acquired aplastic anemia mouse model through the X-ray irradiation in combination with lymphocytes injection. AA Group: the purified Pan T lymphocytes from the spleen of C57BL/6J mice were enriched and injected to the mice through tail vein(5×106), the CB6F1 mice were exposed to 3,4 and 5 Gy X-ray irradiation; TBI Group: the CB6F1 mice were exposed to 3,4 and 5 Gy X-ray irradiation, and were injected with the same volume of PBS buffer; Control group: the CB6F1 mice were only injected with the same volume of PBS buffer. The peripheral blood routine was examined and the number of nucleated cells in bone marrow were calculatedï¼the hematopoiesis changes in bone marrow was examinedï¼flow cytometry was used to examine the distribution of T lymphocytes in bone marrow, and it also used to examine the apoptosis of bone marrow cells and the differentiation of spleen T lymphocytes. RESULTS: Compared with 4, 5 Gy irradiated mice in AA groups, the survival time of 3 Gy irradiated AA groups was significantly prolonged. 3, 4 and 5 Gy X-ray irradiation combined with Pan T lymphocyte injection could successfully induced severe reduction of red blood cells, blood neutrophils, and platelets, severe reduction of bone marrow nucleated cells, severe bone marrow hematopoietic failure, and the significant expansion of T lymphocytes ratio in the bone marrow. CD4+ and CD8+ T cells were both increased, but mainly on CD8+ T cells, and could promote the differentiation of T cells from naïve T cells to effector memory T cells. CONCLUSION: 3, 4 and 5 Gy X-ray irradiation combined with 5×106 pan-T cell injection could successfully induce acquired aplastic anemia through T lymphocyte hyperfunction. Compared with 4, 5 Gy irradiated AA group, the 3 Gy irradiated AA group shows significantly longer survival time, and the peripheral blood routine profile closely resembles the clinical manifestations of AA patients.
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Anemia Aplástica , Animais , Medula Óssea , Células da Medula Óssea , Linfócitos T CD8-Positivos , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Ten-eleven translocation 2 (TET2) functions as a methylcytosine dioxygenase that catalyzes the iterative oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. TET2 has been shown to be crucial for the maintenance and differentiation of hematopoietic stem cells, and its deletion and/or mutations results in the expansion of HSPCs, and leads to hematological malignancies. TET2 mutations were found in a variety of hematological disorders such as CMML (60%), MDS (30%), MPN (13%) and AML (20%). Interestingly, it was shown that CMML patients with TET2 mutation exhibited fewer platelets than CMML patients without TET2 mutation. However, the role and function of TET2 in platelet hemostasis and thrombogenesis is not well defined. Here in this study, using a genetically engineered Tet2 deletion mouse model, we found that the absence of Tet2 caused a decrease in the proportion of MEP cells and hyperploid megakaryocytes. Additionally, Tet2-deficient mice displayed impaired platelet activation and aggregation under stimulation of ADP and low concentrations of thrombin, although the modestly compromised platelet function and MEP differentiation in Tet2-deficient mice could be compensated without affecting blood coagulation function. Our study indicate that Tet2 deficiency leads to mild impairment of platelet function and thrombopoiesis in mice.
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Epigenetic regulations play crucial roles in leukemogenesis and leukemia progression. SUV39H1 is the dominant H3K9 methyltransferase in the hematopoietic system, and its expression declines with aging. However, the role of SUV39H1 via its-mediated repressive modification H3K9me3 in leukemogenesis/leukemia progression remains to be explored. We found that SUV39H1 was down-regulated in a variety of leukemias, including MLL-r AML, as compared with normal individuals. Decreased levels of Suv39h1 expression and genomic H3K9me3 occupancy were observed in LSCs from MLL-r-induced AML mouse models in comparison with that of hematopoietic stem/progenitor cells. Suv39h1 overexpression increased leukemia latency and decreased the frequency of LSCs in MLL-r AML mouse models, while Suv39h1 knockdown accelerated disease progression with increased number of LSCs. Increased Suv39h1 expression led to the inactivation of Hoxb13 and Six1, as well as reversion of Hoxa9/Meis1 downstream target genes, which in turn decelerated leukemia progression. Interestingly, Hoxb13 expression is up-regulated in MLL-AF9-induced AML cells, while knockdown of Hoxb13 in MLL-AF9 leukemic cells significantly prolonged the survival of leukemic mice with reduced LSC frequencies. Our data revealed that SUV39H1 functions as a tumor suppressor in MLL-AF9-induced AML progression. These findings provide the direct link of SUV39H1 to AML development and progression.
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Progressão da Doença , Leucemia Mieloide Aguda/patologia , Metiltransferases/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Repressoras/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Feminino , Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Lisina/metabolismo , Metilação , Camundongos , Transcrição GênicaRESUMO
Soil nitrogen (N) shortage is a problem which affects many developing nations. Crops grown with low soil N levels show a marked decrease in the rate of photosynthesis and this deficiency reduces crop yield significantly. Therefore, developing a better understanding of the mechanisms by which low N levels cause decreased photosynthesis is crucial for maize agriculture. To better understand this process, we assessed the responses of photosynthesis traits and enzymatic activities in the summer maize cultivar Denghai 618 under field conditions with and without the use of N fertilisers. We measured photosynthesis parameters, and compared proteome compositions to identify the mechanisms of physiological and biochemical adaptations to N deficiency in maize. We observed that parameters that indicated the rate of photosynthesis decreased significantly under N deficiency, and this response was associated with leaf senescence. Moreover, we identified 37 proteins involved in leaf photosynthesis, and found that N deficiency significantly affected light-dependent and light-independent reactions in maize leaf photosynthesis. Although further analysis is required to fully elucidate the roles of these proteins in the response to N deficiency, our study identified candidate proteins which may be involved in the regulatory mechanisms involved in reduced photosynthesis under low N conditions in maize.
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Nitrogênio/farmacologia , Fotossíntese/efeitos dos fármacos , Zea mays/fisiologia , Clorofila/metabolismo , Análise por Conglomerados , Eletroforese em Gel Bidimensional , Fluorescência , Nitrogênio/deficiência , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/anatomia & histologia , Folhas de Planta/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Zea mays/efeitos dos fármacosRESUMO
A pot experiment was conducted to study the effects of partial root excision on the growth of two maize cultivars (Zhengdan 958 and Denghai 9) throughout their growth period and the photosynthesis and leaf antioxidant enzyme activities at grain-filling stage under salt stress. Four treatments were installed, i. e., control (no salt), low salt (0.2% NaCl), moderate salt (0.4% NaCl), and high salt (0.6% NaCl). Under low salt stress, the grain yield of Zhengdan 958 and Denghai 9 with partial root excision was increased by 13.1% and 31.4%, respectively, as compared with that of the cultivars with no root excision. At jointing stage, the growth of the cultivars with partial root excision was restrained, the root- and shoot dry masses under the same salt stresses being lesser than those of the cultivars with no root excision, but the growth under the conditions of no salt and low salt recovered quickly. At milk-ripe stage and under no salt and low salt conditions, the root- and shoot dry masses, leaf area, total root length, total root surface area, root activity, leaf chlorophyll content, and ear leaf net photosynthetic rate, stomatal conductance, transpiration rate, and CAT and POD activities of the cultivars with partial root excision were significantly larger than those of the cultivars with no root excision, while the shoot diameter and ear leaf MDA content were in adverse. Moderate and high salt stresses had greater effects on the cultivars with partial root excision. The root- and shoot dry masses, root morphology, and photosynthesis indices of the cultivars with partial root excision were smaller than those of the cultivars with no root excision, so did the grain yields. Throughout the growth period of the cultivars, the growth of the cultivars with partial root excision depended on the salt concentration, i. e., was promoted under no and low salt, and inhibited under moderate and high salt conditions.