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1.
FASEB J ; 38(15): e23870, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-39120151

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) are successfully employed for hematological transplantations, and impaired HSPC function causes hematological diseases and aging. HSPCs maintain the lifelong homeostasis of blood and immune cells through continuous self-renewal and maintenance of the multilineage differentiation potential. TMEM106B is a transmembrane protein localized on lysosomal membranes and associated with neurodegenerative and cardiovascular diseases; however, its roles in HSPCs and hematopoiesis are unknown. Here, we established tmem106bb-/- knockout (KO) zebrafish and showed that tmem106bb KO reduced the proliferation of HSPCs during definitive hematopoiesis. The differentiation potential of HSPCs to lymphoid lineage was reduced, whereas the myeloid and erythroid differentiation potentials of HPSCs were increased in tmem106bb-/- zebrafish. Similar results were obtained with morpholino knockdown of tmem106bb. Mechanistically, TMEM106B interacted with LAMP2A, the lysosomal associated membrane protein 2A, impaired LAMP2A-Cathepsin A interaction, and enhanced LAMP2A stability; tmem106bb KO or TMEM106B knockdown caused LAMP2A degradation and impairment of chaperone-mediated autophagy (CMA). Knockdown of lamp2a caused similar phenotypes to that in tmem106bb-/- zebrafish, and overexpression of lamp2a rescued the impaired phenotypes of HSPCs in tmem106bb-/- embryos. These results uncover a novel molecular mechanism for the maintenance of HSPC proliferation and differentiation through stabilizing LAMP2A via TMEM106B-LAMP2A interaction.


Assuntos
Diferenciação Celular , Proliferação de Células , Células-Tronco Hematopoéticas , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas de Membrana , Peixe-Zebra , Animais , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Lisossomos/metabolismo , Humanos , Hematopoese/fisiologia
2.
EMBO J ; 43(10): 1990-2014, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38605226

RESUMO

Prenatal lethality associated with mouse knockout of Mettl16, a recently identified RNA N6-methyladenosine (m6A) methyltransferase, has hampered characterization of the essential role of METTL16-mediated RNA m6A modification in early embryonic development. Here, using cross-species single-cell RNA sequencing analysis, we found that during early embryonic development, METTL16 is more highly expressed in vertebrate hematopoietic stem and progenitor cells (HSPCs) than other methyltransferases. In Mettl16-deficient zebrafish, proliferation capacity of embryonic HSPCs is compromised due to G1/S cell cycle arrest, an effect whose rescue requires Mettl16 with intact methyltransferase activity. We further identify the cell-cycle transcription factor mybl2b as a directly regulated by Mettl16-mediated m6A modification. Mettl16 deficiency resulted in the destabilization of mybl2b mRNA, likely due to lost binding by the m6A reader Igf2bp1 in vivo. Moreover, we found that the METTL16-m6A-MYBL2-IGF2BP1 axis controlling G1/S progression is conserved in humans. Collectively, our findings elucidate the critical function of METTL16-mediated m6A modification in HSPC cell cycle progression during early embryonic development.


Assuntos
Células-Tronco Hematopoéticas , Metiltransferases , Metilação de RNA , Proteínas de Ligação a RNA , Fatores de Transcrição , Peixe-Zebra , Animais , Humanos , Camundongos , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/genética , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proliferação de Células , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Metiltransferases/metabolismo , Metiltransferases/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Metilação de RNA/genética
3.
Heliyon ; 10(7): e28644, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38586387

RESUMO

Purpose: To investigate the proportion of zonulopathy in patients with age-related cataract, and further explore demographics and ocular characteristics, as well as potential risk factors. Methods: Hospital-based, observational, cross-sectional study. We enrolled consecutive patients who were 45 years or older and diagnosed with age-related cataract and underwent surgery between October 2022 and April 2023 at the Division of Cataract, Beijing Tongren Hospital. Zonulopathy was diagnosed based on intraoperative signs. We calculated the total proportion, age, and gender specific proportions of zonulopathy. We compared the demographic and ocular characteristics between the cases with and without zonulopathy. Univariate and multivariate logistic regression analyses were employed to determine the risk factors associated with the presence of zonulopathy in patients with age-related cataract. Results: A total of 640 age-related cataract patients with a median age of 70.0 (64.0-77.0) were enrolled. Zonulopathy was diagnosed intraoperatively in 70 patients (10.9%). Compared with the patients having no zonulopathy, those with zonulopathy were likely to be older (P < 0.001), have a shallower central ACD (P < 0.001), a thicker lens (P < 0.001) and a shorter AL (P = 0.010). Logistic regression analyses showed that the risk predictors associated with the presence of zonulopathy in patients with age-related cataract were older age (OR, 1.042; P = 0.035) and shallower central ACD (OR, 0.834; P < 0.001). Conclusion: Zonulopathy in age-related cataract patients is not an uncommon finding. Clinicians should be mindful of zonulopathy in patient population with advanced age and shallower ACD.

4.
J Biol Chem ; 300(3): 105772, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38382674

RESUMO

Pre-mRNA splicing is a precise regulated process and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs) and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remains largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In our previous study, we generated a prpf31 knockout (KO) zebrafish line and reported that Prpf31 regulates the survival and differentiation of retinal progenitor cells by modulating the alternative splicing of genes involved in mitosis and DNA repair. In this study, by using the prpf31 KO zebrafish line, we discovered that prpf31 KO zebrafish exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31-deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes.


Assuntos
Fatores de Processamento de RNA , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Desenvolvimento Embrionário , Mutação , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , Células-Tronco/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
5.
Cell Biol Toxicol ; 40(1): 2, 2024 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-38252267

RESUMO

As part of the central nervous system (CNS), the retina senses light and also conducts and processes visual impulses. The damaged development of the retina not only causes visual damage, but also leads to epilepsy, dementia and other brain diseases. Recently, we have reported that copper (Cu) overload induces retinal developmental defects and down-regulates microtubule (MT) genes during zebrafish embryogenesis, but whether the down-regulation of microtubule genes mediates Cu stress induced retinal developmental defects is still unknown. In this study, we found that microtubule gene stmn4 exhibited obviously reduced expression in the retina of Cu overload embryos. Furthermore, stmn4 deficiency (stmn4-/-) resulted in retinal defects similar to those seen in Cu overload embryos, while overexpression of stmn4 effectively rescued retinal defects and cell apoptosis occurred in the Cu overload embryos and larvae. Meanwhile, stmn4 deficient embryos and larvae exhibited reduced mature retinal cells, the down-regulated expression of microtubules and cell cycle-related genes, and the mitotic cell cycle arrests of the retinal cells, which subsequently tended to apoptosis independent on p53. The results of this study demonstrate that Cu stress might lead to retinal developmental defects via down-regulating expression of microtubule gene stmn4, and stmn4 deficiency leads to impaired cell cycle and the accumulation of retinal progenitor cells (RPCs) and their subsequent apoptosis. The study provides a certain referee for copper overload in regulating the retinal development in fish.


Assuntos
Cobre , Retina , Estatmina , Peixe-Zebra , Animais , Apoptose/genética , Ciclo Celular , Cobre/efeitos adversos , Larva , Retina/patologia , Peixe-Zebra/genética , Estatmina/genética , Proteínas de Peixe-Zebra/genética
6.
Front Cell Dev Biol ; 11: 1169941, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37351277

RESUMO

Introduction: Pathogenic mutations in RPGR ORF15, one of two major human RPGR isoforms, were responsible for most X-linked retinitis pigmentosa cases. Previous studies have shown that RPGR plays a critical role in ciliary protein transport. However, the precise mechanisms of disease triggered by RPGR ORF15 mutations have yet to be clearly defined. There are two homologous genes in zebrafish, rpgra and rpgrb. Zebrafish rpgra has a single transcript homologous to human RPGR ORF15; rpgrb has two major transcripts: rpgrb ex1-17 and rpgrb ORF15, similar to human RPGR ex1-19 and RPGR ORF15, respectively. rpgrb knockdown in zebrafish resulted in both abnormal development and increased cell death in the dysplastic retina. However, the impact of knocking down rpgra in zebrafish remains undetermined. Here, we constructed a rpgra mutant zebrafish model to investigate the retina defect and related molecular mechanism. Methods: we utilized transcription activator-like effector nuclease (TALEN) to generate a rpgra mutant zebrafish. Western blot was used to determine protein expression. RT-PCR was used to quantify gene transcription levels. The visual function of embryonic zebrafish was detected by electroretinography. Immunohistochemistry was used to observe the pathological changes in the retina of mutant zebrafish and transmission electron microscope was employed to view subcellular structure of photoreceptor cells. Results: A homozygous rpgra mutant zebrafish with c.1675_1678delins21 mutation was successfully constructed. Despite the normal morphological development of the retina at 5 days post-fertilization, visual dysfunction was observed in the mutant zebrafish. Further histological and immunofluorescence assays indicated that rpgra mutant zebrafish retina photoreceptors progressively began to degenerate at 3-6 months. Additionally, the mislocalization of cone outer segment proteins (Opn1lw and Gnb3) and the accumulation of vacuole-like structures around the connecting cilium below the OSs were observed in mutant zebrafish. Furthermore, Rab8a, a key regulator of opsin-carrier vesicle trafficking, exhibited decreased expression and evident mislocalization in mutant zebrafish. Discussion: This study generated a novel rpgra mutant zebrafish model, which showed retinal degeneration. our data suggested Rpgra is necessary for the ciliary transport of cone-associated proteins, and further investigation is required to determine its function in rods. The rpgra mutant zebrafish constructed in this study may help us gain a better understanding of the molecular mechanism of retinal degeneration caused by RPGR ORF15 mutation and find some useful treatment in the future.

7.
iScience ; 26(4): 106406, 2023 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-37009226

RESUMO

Unbalanced Cu homeostasis has been suggested to be associated with hematopoietic disease, but the roles of Cu overload in the hematopoietic system and the potential mechanisms are obscure. Here, we report a novel association and the novel potential pathways for Cu overload to induce proliferation defects in zebrafish embryonic hematopoietic stem and progenitor cells (HSPCs) via down-regulating expression of foxm1-cytoskeleton axis, which is conserved from fish to mammals. Mechanistically, we show the direct binding of Cu to transcriptional factors HSF1 and SP1 and that Cu overload induces the cytoplasmic aggregation of proteins HSF1 and SP1. These result in the reduced transcriptional activities of HSF1 and SP1 on their downstream FOXM1 as well as the FOXM1 transcriptional activities on cytoskeletons in HSPCs, which leads to ultimately cell proliferation impairment. These findings unveil the novel linkage of Cu overload with specific signaling transduction as well as the subsequent HSPC proliferation defects.

8.
Biochim Biophys Acta Mol Basis Dis ; 1869(4): 166645, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36682603

RESUMO

Retinal photoreceptors execute phototransduction functions and require an efficient system for the transport of materials (e.g. proteins and lipids) from inner segments to outer segments. Cytoplasmic dynein 1 is a minus-end-directed microtubule motor and participates in cargo transport in the cytoplasm. However, the roles of dynein 1 motor in photoreceptor cargo transport and retinal development are still ambiguous. In our present study, the light intermediate chain protein DLIC1 (encoded by dync1li1), links activating adaptors to bind diverse cargos in the dynein 1 motor, was depleted using CRISPR-Cas9 technology in zebrafish. The dync1li1-/- zebrafish displayed progressive degeneration of retinal cone photoreceptors, especially blue cones. The retinal rods were not affected in dync1li1-/- zebrafish. Knockout of DLIC1 resulted in abnormal expression and localization of cone opsins in dync1li1-/- retinas. TUNEL staining suggested that apoptosis was induced after aberrant accumulation of cone opsins in photoreceptors of dync1li1-/- zebrafish. Instead of Rab11 transport, Rab8 transport was disturbed in dync1li1-/- retinas. Our data demonstrate that DLIC1 is required for function maintenance and survival of cone photoreceptors, and hint at an essential role of the cytoplasmic dynein 1 motor in photoreceptor cargo transport.


Assuntos
Opsinas dos Cones , Dineínas do Citoplasma , Células Fotorreceptoras Retinianas Cones , Animais , Opsinas dos Cones/metabolismo , Dineínas do Citoplasma/genética , Dineínas do Citoplasma/metabolismo , Dineínas/genética , Dineínas/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
9.
Obesity (Silver Spring) ; 31(1): 123-138, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36504350

RESUMO

OBJECTIVE: Genetic variants in ninjurin-2 (NINJ2; nerve injury-induced protein 2) confer risk of ischemic strokes and coronary artery disease as well as endothelial activation and inflammation. However, little is known about NINJ2's in vivo functions and underlying mechanisms. METHODS: The phenotypes of NINJ2 knockout mice were analyzed, and mechanisms of NINJ2 that regulate body weight, insulin resistance, and glucose homeostasis and lipogenesis were investigated in vivo and in vitro. RESULTS: This study found that mice lacking NINJ2 showed impaired adipogenesis, increased insulin resistance, and abnormal glucose homeostasis, all of which are risk factors for strokes and coronary artery disease. Mechanistically, NINJ2 directly interacts with insulin receptor/insulin-like growth factor 1 receptor (INSR/IGF1R), and NINJ2 knockdown can block insulin-induced mitotic clonal expansion during preadipocyte differentiation by inhibiting protein kinase B/extracellular signal-regulated kinase (AKT/ERK) signaling and by decreasing the expression of key adipocyte transcriptional regulators CCAAT/enhancer-binding protein ß (C/EBP-ß), C/EBP-α, and peroxisome proliferator-activated receptor γ (PPAR-γ). Furthermore, the interaction between NINJ2 and INSR/IGF1R is needed for maintaining insulin sensitivity in adipocytes and muscle via AKT and glucose transporter type 4. Notably, adenovirus-mediated NINJ2 overexpression can ameliorate diet-induced insulin resistance in mice. CONCLUSIONS: In conclusion, these findings reveal NINJ2 as an important new facilitator of insulin receptors, and the authors propose a unique regulatory mechanism between insulin signaling, adipogenesis, and insulin resistance.


Assuntos
Moléculas de Adesão Celular Neuronais , Resistência à Insulina , Animais , Camundongos , Células 3T3-L1 , Adipogenia/genética , Diferenciação Celular/genética , Doença da Artéria Coronariana , Glucose/metabolismo , Insulina , Resistência à Insulina/genética , PPAR gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Moléculas de Adesão Celular Neuronais/genética
10.
Neurosci Bull ; 39(1): 57-68, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35713844

RESUMO

PiT2 is an inorganic phosphate (Pi) transporter whose mutations are linked to primary familial brain calcification (PFBC). PiT2 mainly consists of two ProDom (PD) domains and a large intracellular loop region (loop7). The PD domains are crucial for the Pi transport, but the role of PiT2-loop7 remains unclear. In PFBC patients, mutations in PiT2-loop7 are mainly nonsense or frameshift mutations that probably cause PFBC due to C-PD1131 deletion. To date, six missense mutations have been identified in PiT2-loop7; however, the mechanisms by which these mutations cause PFBC are poorly understood. Here, we found that the p.T390A and p.S434W mutations in PiT2-loop7 decreased the Pi transport activity and cell surface levels of PiT2. Furthermore, we showed that these two mutations attenuated its membrane localization by affecting adenosine monophosphate-activated protein kinase (AMPK)- or protein kinase B (AKT)-mediated PiT2 phosphorylation. In contrast, the p.S121C and p.S601W mutations in the PD domains did not affect PiT2 phosphorylation but rather impaired its substrate-binding abilities. These results suggested that missense mutations in PiT2-loop7 can cause Pi dyshomeostasis by affecting the phosphorylation-regulated cell-surface localization of PiT2. This study helps understand the pathogenesis of PFBC caused by PiT2-loop7 missense mutations and indicates that increasing the phosphorylation levels of PiT2-loop7 could be a promising strategy for developing PFBC therapies.


Assuntos
Mutação de Sentido Incorreto , Fosfatos , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III , Humanos , Membrana Celular , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética
11.
Development ; 149(17)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35929537

RESUMO

Mutations that occur in RNA-splicing machinery may contribute to hematopoiesis-related diseases. How splicing factor mutations perturb hematopoiesis, especially in the differentiation of erythro-myeloid progenitors (EMPs), remains elusive. Dhx38 is a pre-mRNA splicing-related DEAH box RNA helicase, for which the physiological functions and splicing mechanisms during hematopoiesis currently remain unclear. Here, we report that Dhx38 exerts a broad effect on definitive EMPs as well as the differentiation and maintenance of hematopoietic stem and progenitor cells (HSPCs). In dhx38 knockout zebrafish, EMPs and HSPCs were found to be arrested in mitotic prometaphase, accompanied by a 'grape' karyotype, owing to the defects in chromosome alignment. Abnormal alternatively spliced genes related to chromosome segregation, the microtubule cytoskeleton, cell cycle kinases and DNA damage were present in the dhx38 mutants. Subsequently, EMPs and HSPCs in dhx38 mutants underwent P53-dependent apoptosis. This study provides novel insights into alternative splicing regulated by Dhx38, a process that plays a crucial role in the proliferation and differentiation of fetal EMPs and HSPCs.


Assuntos
Processamento Alternativo , Peixe-Zebra , Processamento Alternativo/genética , Animais , Hematopoese/genética , Células-Tronco Hematopoéticas , Células Progenitoras Mieloides , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
12.
Biochim Biophys Acta Mol Basis Dis ; 1867(11): 166233, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34339841

RESUMO

Genetic mutations in heat shock factor 4 (Hsf4) is associated with both congenital and age-related cataracts. Hsf4 regulates lens development through its ability to both activate and inhibit transcription. Previous studies suggested Hsf4 is involved in modulating cellular senescence depending on p21cip1 and p27 kip1 expression in MEF cells. Here, we found that Hsf4 acts as a suppressor of p21cip1 expression and plays an anti-senescence role during lens development. Knocking out Hsf4 facilitated UVB-induced cellular senescence in mouse lens epithelial cells (mLECs). p21cip1 was upregulated at both the mRNA and protein levels in HSF4-/- mLECs under control and UVB-treated conditions, and knockdown of p21cip1 by siRNA alleviated UVB-induced cellular senescence. HSF4 directly bound to the p21cip1 promoter and increased H3K27m3 levels at the p21cip1 proximal promoter region by recruiting the methyltransferase EZH2. In animal models, p21cip1 was gradually upregulated in wild-type mouse lenses with increasing age, while Hsf4 levels decreased. We generated a Hsf4 mutant mice line (Hsf4del-42) which displayed obvious congenital cataract phenotype. The expression of p21cip1 and senescence-associated cytokines were induced in the cataractous lenses of Hsf4del-42 mice. H3K27m3 and EZH2 levels decreased in p21cip1 promoters in the lenses of Hsf4del-42 mice. The SA-ß-Gal activities were positive in lens epithelia of aged Hsf4null zebrafish compared to wild-type lenses. p21cip1 and senescence-associated cytokines levels were also upregulated in lenses of Hsf4null zebrafish. Accordingly, we propose that HSF4 plays a protective role in lens epithelial cells against cellular senescence during lens development and aging, partly by fine-tuning p21cip1 expression.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Fatores de Transcrição de Choque Térmico/deficiência , Cristalino/patologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Envelhecimento/genética , Animais , Animais Geneticamente Modificados , Catarata/genética , Catarata/patologia , Linhagem Celular , Senescência Celular/genética , Senescência Celular/efeitos da radiação , Metilação de DNA , Modelos Animais de Doenças , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Fatores de Transcrição de Choque Térmico/genética , Histonas/genética , Histonas/metabolismo , Humanos , Cristalino/citologia , Cristalino/crescimento & desenvolvimento , Cristalino/efeitos da radiação , Camundongos , Regiões Promotoras Genéticas , Raios Ultravioleta/efeitos adversos , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
13.
Brief Bioinform ; 22(5)2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-33822848

RESUMO

Irregular splicing was associated with tumor formation and progression in renal cell carcinoma (RCC) and many other cancers. By using splicing data in the TCGA SpliceSeq database, RCC subtype classification was performed and splicing features and their correlations with clinical course, genetic variants, splicing factors, pathways activation and immune heterogeneity were systemically analyzed. In this research, alternative splicing was found useful for classifying RCC subtypes. Splicing inefficiency with upregulated intron retention and cassette exon was associated with advanced conditions and unfavorable overall survival of patients with RCC. Splicing characteristics like splice site strength, guanine and cytosine content and exon length may be important factors disrupting splicing balance in RCC. Other than cis-acting and trans-acting regulation, alternative splicing also differed in races and tissue types and is also affected by mutation conditions, pathway settings and the response to environmental changes. Severe irregular splicing in tumor not only indicated terrible intra-cellular homeostasis, but also changed the activity of cancer-associated pathways by different splicing effects including isoforms switching and expression regulation. Moreover, irregular splicing and splicing-associated antigens were involved in immune reprograming and formation of immunosuppressive tumor microenvironment. Overall, we have described several clinical and molecular features in RCC splicing subtypes, which may be important for patient management and targeting treatment.


Assuntos
Processamento Alternativo , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Mutação , Carcinoma de Células Renais/classificação , Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/classificação , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Microambiente Tumoral/genética
14.
Nucleic Acids Res ; 49(4): 2027-2043, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33476374

RESUMO

Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5' splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis.


Assuntos
Processamento Alternativo , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Retina/embriologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Apoptose , Sistemas CRISPR-Cas , Sobrevivência Celular , Dano ao DNA , Reparo do DNA , Éxons , Técnicas de Inativação de Genes , Pontos de Checagem da Fase M do Ciclo Celular , Células-Tronco Neurais/citologia , Neurônios Retinianos/citologia , Neurônios Retinianos/metabolismo , Fuso Acromático/ultraestrutura , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
15.
Mol Vis ; 26: 670-678, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33088171

RESUMO

Purpose: To identify the genetic cause in a four-generation Chinese family with Axenfeld-Rieger syndrome (ARS). Methods: The family members received clinical examinations of the eye, tooth, periumbilical skin, and heart. Sanger sequencing and whole-exome sequencing (WES) were performed to screen potential mutations. The genomic deletion region around the PITX2 gene was estimated from single nucleotide polymorphism (SNP) data from WES and then confirmed with "quantitative PCR (qPCR) using a set of primers. The DNA breakpoint was further identified with long-range PCR and Sanger sequencing. Results: Symptoms including anterior segment dysplasia of the eye (iris dysplasia, multiple pupils, and posterior embryotoxon), dental dysplasia, and periumbilical skin redundancy were present in all of the affected individuals. Three of them had glaucoma. Corneal abnormalities (inferior sclerocornea, corneal endothelial dystrophy, and central corneal scar) were seen in most of the affected individuals. Cataract, limited eye movement, electrocardiographic abnormalities, intellectual disability, and recurrent miscarriages were observed in some of the affected individuals. No mutations in the coding and exon-intron adjacent regions of the PITX2 and FOXC1 genes were identified with Sanger sequencing. According to the SNP data from WES, we suspected that there might be a deletion region (at most 1.6 Mb) around the PITX2 gene. With the use of qPCR and long-range PCR, we identified a 53,840 bp deletion (chr4: 111,535,454-111,588,933) spanning PITX2 and PANCR. The genomic deletion cosegregated with the major ARS symptoms observed in the family members. Conclusions: With the help of WES, qPCR, and long-range PCR, we identified a genomic deletion encompassing PITX2 and the adjacent noncoding gene PANCR in a Chinese family with ARS. The clinical features of the affected individuals are reported. This work may broaden understanding of the phenotypic and mutational spectrums related to ARS.


Assuntos
Segmento Anterior do Olho/anormalidades , Anormalidades do Olho/genética , Oftalmopatias Hereditárias/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Adulto , Segmento Anterior do Olho/fisiopatologia , Povo Asiático , Eletrocardiografia , Anormalidades do Olho/fisiopatologia , Oftalmopatias Hereditárias/fisiopatologia , Feminino , Fatores de Transcrição Forkhead/genética , Genótipo , Glaucoma/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Deleção de Sequência , Sequenciamento do Exoma , Proteína Homeobox PITX2
16.
FASEB J ; 34(9): 11997-12008, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32738093

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) have the ability to self-renew and differentiate into various blood cells, thus playing an important role in maintenance of lifelong hematopoiesis. Brahma-related gene 1 (BRG1), which acts as the ATP subunit of mammalian SWI-SNF-related chromatin remodeling complexes, is involved in human acute myeloid leukemia and highly expresses in short-term HSPCs. But its role and regulatory mechanism for HSPC development have not yet been well established. Here, we generated a brg1 knockout zebrafish model using TALEN technology. We found that in brg1-/- embryo, the primitive hematopoiesis remained well, while definitive hematopoiesis formation was significantly impaired. The number of hemogenic endothelial cells was decreased, further affecting definitive hematopoiesis with reduced myeloid and lymphoid cells. During embryogenesis, the nitric oxide (NO) microenvironment in brg1-/- embryo was seriously damaged and the reduction of HSPCs could be partially rescued by a NO donor. Chromatin immunoprecipitation (ChIP) assays showed that BRG1 could bind to the promoter of KLF2 and trigger its transcriptional activity of NO synthase. Our findings show that Brg1 promotes klf2a expression in hemogenic endothelium and highlight a novel mechanism for HSPC formation and maintenance.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Embrião não Mamífero/embriologia , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Nicho de Células-Tronco , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/citologia , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Elementos de Resposta , Transcrição Gênica , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genética
17.
Aging (Albany NY) ; 12(13): 13594-13617, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32554860

RESUMO

The general transcription factor, CREB has been shown to play an essential role in promoting cell proliferation, neuronal survival and synaptic plasticity in the nervous system. However, its function in stress response remains to be elusive. In the present study, we demonstrated that CREB plays a major role in mediating stress response. In both rat lens organ culture and mouse lens epithelial cells (MLECs), CREB promotes oxidative stress-induced apoptosis. To confirm that CREB is a major player mediating the above stress response, we established stable lines of MLECs stably expressing CREB and found that they are also very sensitive to oxidative stress-induced apoptosis. To define the underlying mechanism, RNAseq analysis was conducted. It was found that CREB significantly suppressed expression of the αB-crystallin gene to sensitize CREB-expressing cells undergoing oxidative stress-induced apoptosis. CREB knockdown via CRISPR/CAS9 technology led to upregulation of αB-crystallin and enhanced resistance against oxidative stress-induced apoptosis. Moreover, overexpression of exogenous human αB-crystallin can restore the resistance against oxidative stress-induced apoptosis. Finally, we provided first evidence that CREB directly regulates αB-crystallin gene. Together, our results demonstrate that CREB is an important transcription factor mediating stress response, and it promotes oxidative stress-induced apoptosis by suppressing αB-crystallin expression.


Assuntos
Cristalinas/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Estresse Oxidativo/genética , Cadeia B de alfa-Cristalina/genética , Animais , Apoptose/genética , Catarata/genética , Catarata/patologia , Linhagem Celular , Sobrevivência Celular/genética , Células Cultivadas , Regulação para Baixo , Células Epiteliais , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Cristalino/citologia , Cristalino/patologia , Masculino , Camundongos , Técnicas de Cultura de Órgãos , RNA-Seq , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Regulação para Cima , Cadeia B de alfa-Cristalina/metabolismo
18.
Biochim Biophys Acta Mol Basis Dis ; 1866(5): 165724, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32061775

RESUMO

Removal of nuclei in lens fiber cells is required for organelle-free zone (OFZ) formation during lens development. Defect in degradation of nuclear DNA leads to cataract formation. DNase2ß degrades nuclear DNA of lens fiber cells during lens differentiation in mouse. Hsf4 is the principal heat shock transcription factor in lens and facilitates the lens differentiation. Knockout of Hsf4 in mouse and zebrafish resulted in lens developmental defect that was characterized by retaining of nuclei in lens fiber cells. In previous in vitro studies, we found that Hsf4 promoted DNase2ß expression in human and mouse lens epithelial cells. In this study, it was found that, instead of DNase2ß, DNase1l1l is uniquely expressed in zebrafish lens and was absent in Hsf4-/- zebrafish lens. Using CRISPR-Cas9 technology, a DNase1l1l knockout zebrafish line was constructed, which developed cataract. Deletion of DNase1l1l totally abrogated lens primary and secondary fiber cell denucleation process, whereas had little effect on the clearance of other organelles. The transcriptional regulation of DNase1l1l was dramatically impaired in Hsf4-/- zebrafish lens. Rescue of DNase1l1l mRNA into Hsf4-/- zebrafish embryos alleviated its defect in lens fiber cell denucleation. Our results in vivo demonstrated that DNase1l1l is the primary DNase responsible for nuclear DNA degradation in lens fiber cells, and Hsf4 can transcriptionally activate DNase1l1l expression in zebrafish.


Assuntos
Catarata/genética , Desoxirribonucleases/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição de Choque Térmico/metabolismo , Cristalino/embriologia , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Catarata/patologia , Núcleo Celular/metabolismo , Desoxirribonucleases/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Técnicas de Inativação de Genes , Fatores de Transcrição de Choque Térmico/genética , Humanos , Cristalino/citologia , Cristalino/metabolismo , Cristalino/patologia , Masculino , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
19.
Biochim Biophys Acta Gen Subj ; 1864(3): 129496, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31786107

RESUMO

BACKGROUND: Germline mutations in heat shock factor 4 (HSF4) cause congenital cataracts. Previously, we have shown that HSF4 is involved in regulating lysosomal pH in mouse lens epithelial cell in vitro. However, the underlying mechanism remains unclear. METHODS: HSF4-deficient mouse lens epithelial cell lines and zebrafish were used in this study. Immunoblotting and quantitative RT-PCR were used for expression analysis. The protein-protein interactions were tested with GST-pull downs. The lysosomes were fractioned by ultracentrifugation. RESULTS: HSF4 deficiency or knock down of αB-crystallin elevates lysosomal pH and increases the ubiquitination and degradation of ATP6V1A by the proteasome. αB-crystallin localizes partially in the lysosome and interacts solely with the ATP6V1A protein of the V1 complex of V-ATPase. Furthermore, αB-crystallin can co-precipitate with mTORC1 and ATP6V1A in GST pull down assays. Inhibition of mTORC1 by rapamycin or siRNA can lead to dissociation of αB-crystallin from the ATP6V1A and mTORC1complex, shortening the half-life of ATP6V1A and increasing the lysosomal pH. Mutation of ATP6V1A/S441A (the predicted mTOR phosphorylation site) reduces its association with αB-crystallin. In the zebrafish model, HSF4 deficiency reduces αB-crystallin expression and elevates the lysosomal pH in lens tissues. CONCLUSION: HSF4 regulates lysosomal acidification by controlling the association of αB-crystallin with ATP6V1A and mTOR and regulating ATP6V1A protein stabilization. GENERAL SIGNIFICANCE: This study uncovers a novel function of αB-crystallin, demonstrating that αB-crystallin can regulate lysosomal ATP6V1A protein stabilization by complexing to ATP6V1A and mTOR. This highlights a novel mechanism by which HSF4 regulates the proteolytic process of organelles during lens development.


Assuntos
Fatores de Transcrição de Choque Térmico/metabolismo , Lisossomos/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Animais , Linhagem Celular , Cristalinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Humanos , Cristalino/metabolismo , Lisossomos/fisiologia , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitinação , ATPases Vacuolares Próton-Translocadoras/metabolismo , Peixe-Zebra/metabolismo
20.
Biochim Biophys Acta Mol Basis Dis ; 1865(10): 2694-2705, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31348989

RESUMO

Leber congenital amaurosis (LCA) is the most serious form of inherited retinal dystrophy that leads to blindness or severe visual impairment within a few months after birth. Approximately 1-2% of the reported cases are caused by mutations in the LCA5 gene. This gene encodes a ciliary protein called LCA5 that is localized to the connecting cilium of photoreceptors. The retinal phenotypes caused by LCA5 mutations and the underlying pathological mechanisms are still not well understood. In this study, we knocked out the lca5 gene in zebrafish using CRISPR/Cas9 technology. An early onset visual defect is detected by the ERG in 7 dpf lca5-/- zebrafish. Histological analysis by HE staining and immunofluorescence reveal progressive degeneration of rod and cone photoreceptors, with a pattern that cones are more severely affected than rods. In addition, ultrastructural analysis by transmission electron microscopy shows disordered and broken membrane discs in rods' and cones' outer segments, respectively. In our lca5-/- zebrafish, the red-cone opsin and cone α-transducin are selectively mislocalized to the inner segment and synaptic terminal. Moreover, we found that Ift88, a key component of the intraflagellar transport complex, is retained in the outer segments. These data suggest that the intraflagellar transport complex-mediated outer segment protein trafficking might be impaired due to lca5 deletion, which finally leads to a type of retinal degeneration mimicking the phenotype of cone-rod dystrophy in human. Our work provides a novel animal model to study the physiological function of LCA5 and develop potential treatments of LCA.


Assuntos
Distrofias de Cones e Bastonetes/genética , Predisposição Genética para Doença/genética , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/metabolismo , Transporte Proteico/fisiologia , Peixe-Zebra/genética , Animais , Sistemas CRISPR-Cas , Cílios/metabolismo , Modelos Animais de Doenças , Proteínas do Olho/metabolismo , Técnicas de Inativação de Genes , Humanos , Amaurose Congênita de Leber/patologia , Proteínas Associadas aos Microtúbulos , Fenótipo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
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