RESUMO
ABSTRACT: Transient abnormal myelopoiesis (TAM) is a common complication in newborns with Down syndrome (DS). It commonly progresses to myeloid leukemia (ML-DS) after spontaneous regression. In contrast to the favorable prognosis of primary ML-DS, patients with refractory/relapsed ML-DS have poor outcomes. However, the molecular basis for refractoriness and relapse and the full spectrum of driver mutations in ML-DS remain largely unknown. We conducted a genomic profiling study of 143 TAM, 204 ML-DS, and 34 non-DS acute megakaryoblastic leukemia cases, including 39 ML-DS cases analyzed by exome sequencing. Sixteen novel mutational targets were identified in ML-DS samples. Of these, inactivations of IRX1 (16.2%) and ZBTB7A (13.2%) were commonly implicated in the upregulation of the MYC pathway and were potential targets for ML-DS treatment with bromodomain-containing protein 4 inhibitors. Partial tandem duplications of RUNX1 on chromosome 21 were also found, specifically in ML-DS samples (13.7%), presenting its essential role in DS leukemia progression. Finally, in 177 patients with ML-DS treated following the same ML-DS protocol (the Japanese Pediatric Leukemia and Lymphoma Study Group acute myeloid leukemia -D05/D11), CDKN2A, TP53, ZBTB7A, and JAK2 alterations were associated with a poor prognosis. Patients with CDKN2A deletions (n = 7) or TP53 mutations (n = 4) had substantially lower 3-year event-free survival (28.6% vs 90.5%; P < .001; 25.0% vs 89.5%; P < .001) than those without these mutations. These findings considerably change the mutational landscape of ML-DS, provide new insights into the mechanisms of progression from TAM to ML-DS, and help identify new therapeutic targets and strategies for ML-DS.
Assuntos
Síndrome de Down , Mutação , Humanos , Síndrome de Down/genética , Síndrome de Down/complicações , Masculino , Feminino , Reação Leucemoide/genética , Lactente , Pré-Escolar , Sequenciamento do Exoma , Prognóstico , Leucemia Mieloide/genética , Recém-Nascido , Criança , Subunidade alfa 2 de Fator de Ligação ao Core/genéticaRESUMO
Inherited bone-marrow-failure syndromes (IBMFSs) include heterogeneous genetic disorders characterized by bone-marrow failure, congenital anomalies, and an increased risk of malignancy. Many lines of evidence have suggested that p53 activation might be central to the pathogenesis of IBMFSs, including Diamond-Blackfan anemia (DBA) and dyskeratosis congenita (DC). However, the exact role of p53 activation in each clinical feature remains unknown. Here, we report unique de novo TP53 germline variants found in two individuals with an IBMFS accompanied by hypogammaglobulinemia, growth retardation, and microcephaly mimicking DBA and DC. TP53 is a tumor-suppressor gene most frequently mutated in human cancers, and occasional germline variants occur in Li-Fraumeni cancer-predisposition syndrome. Most of these mutations affect the core DNA-binding domain, leading to compromised transcriptional activities. In contrast, the variants found in the two individuals studied here caused the same truncation of the protein, resulting in the loss of 32 residues from the C-terminal domain (CTD). Unexpectedly, the p53 mutant had augmented transcriptional activities, an observation not previously described in humans. When we expressed this mutant in zebrafish and human-induced pluripotent stem cells, we observed impaired erythrocyte production. These findings together with close similarities to published knock-in mouse models of TP53 lacking the CTD demonstrate that the CTD-truncation mutations of TP53 cause IBMFS, providing important insights into the previously postulated connection between p53 and IBMFSs.
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Doenças da Medula Óssea/genética , Medula Óssea/patologia , Células Germinativas/patologia , Mutação/genética , Proteína Supressora de Tumor p53/genética , Adolescente , Adulto , Agamaglobulinemia/genética , Anemia de Diamond-Blackfan/genética , Animais , Pré-Escolar , Eritrócitos/patologia , Feminino , Transtornos do Crescimento/genética , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Lactente , Recém-Nascido , Masculino , Camundongos , Pessoa de Meia-Idade , Adulto Jovem , Peixe-ZebraRESUMO
Human telomeric RNA has been identified as a key component of the telomere machinery. Recently, the growing evidence suggests that the telomeric RNA forms G-quadruplex structures to play an important role in telomere protection and regulation. In the present studies, we developed a 19F NMR spectroscopy method to investigate the telomeric RNA G-quadruplex structures in vitro and in living cells. We demonstrated that the simplicity and sensitivity of 19F NMR approach can be used to directly observe the dimeric and two-subunits stacked G-quadruplexes in vitro and in living cells and quantitatively characterize the thermodynamic properties of the G-quadruplexes. By employing the 19F NMR in living cell experiment, we confirmed for the first time that the higher-order G-quadruplex exists in cells. We further demonstrated that telomere RNA G-quadruplexes are converted to the higher-order G-quadruplex under molecular crowding condition, a cell-like environment. We also show that the higher-order G-quadruplex has high thermal stability in crowded solutions. The finding provides new insight into the structural behavior of telomere RNA G-quadruplex in living cells. These results open new avenues for the investigation of G-quadruplex structures in vitro and in living cells.
Assuntos
Quadruplex G , Espectroscopia de Ressonância Magnética , RNA/química , Telômero/química , Animais , Sequência de Bases , Sobrevivência Celular , Flúor , Humanos , Substâncias Macromoleculares/metabolismo , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Temperatura , Termodinâmica , Xenopus laevisRESUMO
RNAcentral is a database of non-coding RNA (ncRNA) sequences that aggregates data from specialised ncRNA resources and provides a single entry point for accessing ncRNA sequences of all ncRNA types from all organisms. Since its launch in 2014, RNAcentral has integrated twelve new resources, taking the total number of collaborating database to 22, and began importing new types of data, such as modified nucleotides from MODOMICS and PDB. We created new species-specific identifiers that refer to unique RNA sequences within a context of single species. The website has been subject to continuous improvements focusing on text and sequence similarity searches as well as genome browsing functionality. All RNAcentral data is provided for free and is available for browsing, bulk downloads, and programmatic access at http://rnacentral.org/.
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Bases de Dados de Ácidos Nucleicos , RNA não Traduzido/química , Animais , Genômica , Humanos , Nucleotídeos/química , Análise de Sequência de RNA , Especificidade da EspécieRESUMO
Mutations in genes encoding ribosomal proteins have been identified in Diamond-Blackfan anemia (DBA), a rare genetic disorder that presents with a prominent erythroid phenotype. TP53 has been implicated in the pathophysiology of DBA with ribosomal protein (RP) L11 playing a crucial role in the TP53 response. Interestingly, RPL11 also controls the transcriptional activity of c-Myc, an oncoprotein that positively regulates ribosome biogenesis. In the present study, we analyzed the consequences of rpl11 depletion on erythropoiesis and ribosome biogenesis in zebrafish. As expected, Rpl11-deficient zebrafish exhibited defects in ribosome biogenesis and an anemia phenotype. However, co-inhibition of Tp53 did not alleviate the erythroid aplasia in these fish. Next, we explored the role of c-Myc in RPL11-deficient cellular and animal models. c-Myc and its target nucleolar proteins showed upregulation and increased localization in the head region of Rpl11-deficient zebrafish, where the morphological abnormalities and tp53 expression were more pronounced. Interestingly, in blood cells derived from DBA patients with mutations in RPL11, the biogenesis of ribosomes was defective, but the expression level of c-Myc and its target nucleolar proteins was unchanged. The results suggest a model whereby RPL11 deficiency activates the synthesis of c-Myc target nucleolar proteins, which subsequently triggers a p53 response. These results further demonstrate that the induction of Tp53 mediates the morphological, but not erythroid, defects associated with RPL11 deficiency.
Assuntos
Anemia de Diamond-Blackfan/fisiopatologia , Proteínas Ribossômicas/deficiência , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patologia , Animais , Modelos Animais de Doenças , Eritropoese/genética , Proteínas de Peixes/deficiência , Proteínas de Peixes/genética , Genes myc , Genes p53 , Humanos , Mutação , Processamento Pós-Transcricional do RNA , Proteínas Ribossômicas/genética , Peixe-ZebraRESUMO
Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder caused by survival motor neuron gene mutations. Variant forms of SMA accompanied by additional clinical presentations have been classified as atypical SMA and are thought to be caused by variants in as yet unidentified causative genes. Here, we presented the clinical findings of two siblings with an SMA variant followed by progressive cerebral atrophy, and the results of whole-exome sequencing analyses of the family quartet that was performed to identify potential causative variants. We identified two candidate homozygous missense variants, R942Q in the tubulin-folding cofactor D (TBCD) gene and H250Q in the bromo-adjacent homology domain and coiled-coil containing 1 (BAHCC1) gene, located on chromosome 17q25.3 with an interval of 1.4 Mbp. The in silico analysis of both variants suggested that TBCD rather than BAHCC1 was likely the pathogenic gene (TBCD sensitivity, 0.68; specificity, 0.97; BAHCC1 sensitivity, 1.00; specificity, 0.00). Thus, our results show that TBCD is a likely novel candidate gene for atypical SMA with progressive cerebral atrophy. TBCD is predicted to have important functions on tubulin integrity in motor neurons as well as in the central nervous system.
Assuntos
Encefalopatias/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas/genética , Atrofias Musculares Espinais da Infância/genética , Encefalopatias/fisiopatologia , Criança , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Homozigoto , Humanos , Neurônios Motores/patologia , Mutação de Sentido Incorreto , Linhagem , Atrofias Musculares Espinais da Infância/fisiopatologiaRESUMO
Alternative splicing (AS) plays an important role in regulating mammalian heart development, but a link between misregulated splicing and congenital heart defects (CHDs) has not been shown. We reported that more than 50% of genes associated with heart development were alternatively spliced in the right ventricle (RV) of infants with tetralogy of Fallot (TOF). Moreover, there was a significant decrease in the level of 12 small cajal body-specific RNAs (scaRNAs) that direct the biochemical modification of specific nucleotides in spliceosomal RNAs. We sought to determine if scaRNA levels influence patterns of AS and heart development. We used primary cells derived from the RV of infants with TOF to show a direct link between scaRNA levels and splice isoforms of several genes that regulate heart development (e.g., GATA4, NOTCH2, DAAM1, DICER1, MBNL1 and MBNL2). In addition, we used antisense morpholinos to knock down the expression of two scaRNAs (scarna1 and snord94) in zebrafish and saw a corresponding disruption of heart development with an accompanying alteration in splice isoforms of cardiac regulatory genes. Based on these combined results, we hypothesize that scaRNA modification of spliceosomal RNAs assists in fine tuning the spliceosome for dynamic selection of mRNA splice isoforms. Our results are consistent with disruption of splicing patterns during early embryonic development leading to insufficient communication between the first and second heart fields, resulting in conotruncal misalignment and TOF. Our findings represent a new paradigm for determining the mechanisms underlying congenital cardiac malformations.
Assuntos
Processamento Alternativo/genética , Corpos Enovelados/genética , Coração/embriologia , Coração/crescimento & desenvolvimento , MicroRNAs/fisiologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/genética , Humanos , Lactente , Recém-Nascido , Vertebrados/embriologia , Vertebrados/genética , Vertebrados/crescimento & desenvolvimento , Peixe-ZebraRESUMO
Diamond-Blackfan anaemia is a congenital bone marrow failure syndrome that is characterized by red blood cell aplasia. The disease has been associated with mutations or large deletions in 11 ribosomal protein genes including RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS29, RPL5, RPL11, RPL26 and RPL35A as well as GATA1 in more than 50% of patients. However, the molecular aetiology of many Diamond-Blackfan anaemia cases remains to be uncovered. To identify new mutations responsible for Diamond-Blackfan anaemia, we performed whole-exome sequencing analysis of 48 patients with no documented mutations/deletions involving known Diamond-Blackfan anaemia genes except for RPS7, RPL26, RPS29 and GATA1. Here, we identified a de novo splicing error mutation in RPL27 and frameshift deletion in RPS27 in sporadic patients with Diamond-Blackfan anaemia. In vitro knockdown of gene expression disturbed pre-ribosomal RNA processing. Zebrafish models of rpl27 and rps27 mutations showed impairments of erythrocyte production and tail and/or brain development. Additional novel mutations were found in eight patients, including RPL3L, RPL6, RPL7L1T, RPL8, RPL13, RPL14, RPL18A and RPL31. In conclusion, we identified novel germline mutations of two ribosomal protein genes responsible for Diamond-Blackfan anaemia, further confirming the concept that mutations in ribosomal protein genes lead to Diamond-Blackfan anaemia.
Assuntos
Anemia de Diamond-Blackfan/genética , Mutação em Linhagem Germinativa , Metaloproteínas/genética , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Anemia de Diamond-Blackfan/fisiopatologia , Animais , Pré-Escolar , Análise Mutacional de DNA/métodos , Eritropoese/genética , Exoma/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem , RNA Ribossômico/genética , Peixe-ZebraRESUMO
Poikiloderma with neutropenia (PN) is a rare inherited disorder characterized by poikiloderma, facial dysmorphism, pachyonychia, short stature and neutropenia. The molecular testing of PN patients has identified mutations in the C16orf57 gene, which encodes a protein referred to as USB1 (U Six Biogenesis 1). In this study, we developed a zebrafish model of PN by the microinjection of morpholino antisense oligos to suppress usb1 gene function. Severe morphological defects, including a bent tail, thin yolk extension and reduced body length, were predominant in the Usb1-suppressed embryos (morphants). We also observed significantly decreased number of neutrophils in the morphants by Sudan Black staining. Interestingly, the splicing of genes involved in neutrophil differentiation and development, such as mpx, ncf1, ela3l and npsn, was aberrant in the morphants. However, the splicing of haematopoietic precursors and erythroid-specific genes was unaltered. Importantly, the neutrophil defects were almost completely rescued by co-injection of ela3l mRNA, the most markedly affected gene in the morphants. Our study demonstrated a possible role of USB1 in modulating the tissue-specific gene splicing that eventually leads to the impaired development of neutrophils. This zebrafish model could serve as a valuable tool to investigate the causative role of USB1 in PN pathogenesis.
Assuntos
Diferenciação Celular/genética , Exorribonucleases/genética , Neutropenia/genética , Neutropenia/patologia , Neutrófilos/metabolismo , Splicing de RNA , Anormalidades da Pele/genética , Anormalidades da Pele/patologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Modelos Animais de Doenças , Exorribonucleases/metabolismo , Técnicas de Silenciamento de Genes , Morfolinos/metabolismo , Neutropenia/metabolismo , Neutrófilos/patologia , Oligonucleotídeos Antissenso/metabolismo , Anormalidades da Pele/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Protein-RNA interactions are essential for many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the protein surface shape (dented, intermediate or protruded) and the RNA base pairing properties (paired or unpaired nucleotides) at the interfaces of 91 protein-RNA complexes derived from the Protein Data Bank. Dented protein surfaces prefer unpaired nucleotides to paired ones at the interface, and hydrogen bonds frequently occur between the protein backbone and RNA bases. In contrast, protruded protein surfaces do not show such a preference, rather, electrostatic interactions initiate the formation of hydrogen bonds between positively charged amino acids and RNA phosphate groups. Interestingly, in many protein-RNA complexes that interact via an RNA loop, an aspartic acid is favored at the interface. Moreover, in most of these complexes, nucleotide bases in the RNA loop are flipped out and form hydrogen bonds with the protein, which suggests that aspartic acid is important for RNA loop recognition through a base-flipping process. This study provides fundamental insights into the role of the shape of the protein surface and RNA secondary structures in mediating protein-RNA interactions.
Assuntos
Proteínas de Ligação a RNA/química , RNA/química , Aminoácidos/química , Pareamento de Bases , Ligação de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Propriedades de SuperfícieRESUMO
Non-coding RNAs (ncRNAs) play key roles in diverse cellular activities, and efficient ncRNA function requires extensive posttranscriptional nucleotide modifications. Small nucleolar RNAs (snoRNAs) are a group of ncRNAs that guide the modification of specific nucleotides in ribosomal RNAs (rRNAs) and small nuclear RNAs. To investigate the physiological relevance of rRNA modification in vertebrates, we suppressed the expression of three snoRNAs (U26, U44 and U78), either by disrupting the host gene splicing or by inhibiting the snoRNA precursor processing, and analyzed the consequences of snoRNA loss-of-function in zebrafish. Using a highly sensitive mass spectrometric analysis, we found that decreased snoRNA expression reduces the snoRNA-guided methylation of the target nucleotides. Impaired rRNA modification, even at a single site, led to severe morphological defects and embryonic lethality in zebrafish, which suggests that rRNA modifications play an essential role in vertebrate development. This study highlights the importance of posttranscriptional modifications and their role in ncRNA function in higher eukaryotes.
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Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Animais , Metilação , Morfolinos , RNA Nucleolar Pequeno/antagonistas & inibidores , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
The recently identified molecule aquaporin-11 (AQP11) has a unique amino acid sequence pattern that includes an Asn-Pro-Cys (NPC) motif, corresponding to the N-terminal Asn-Pro-Ala (NPA) signature motif of conventional AQPs. In this study, we examined the effect of the mutation of the NPC motif on the subcellular localization, oligomerization, and water permeability of AQP11 in transfected mammalian cells. Furthermore, the effect was also assessed using zebrafish. Site-directed mutation at the NPC motif did not affect the subcellular localization of AQP11 but reduced its oligomerization. A cell swelling assay revealed that cells expressing AQP11 with a mutated NPC motif had significantly lower osmotic water permeability than cells expressing wild-type AQP11. Zebrafish deficient in endogenous AQP11 showed a deformity in the tail region at an early stage of development. This phenotype was dramatically rescued by injection of human wild-type AQP11 mRNA, whereas the effect of mRNA for AQP11 with a mutated NPC motif was less marked. Although the NPA motif is known to be important for formation of water-permeable pores by conventional AQPs, our observations suggest that the corresponding NPC motif of AQP11 is essential for full expression of molecular function.
Assuntos
Aquaporinas/genética , Dipeptídeos , Mutação , Motivos de Aminoácidos/genética , Animais , Aquaporinas/administração & dosagem , Aquaporinas/farmacologia , Aquaporinas/fisiologia , Células CHO , Permeabilidade da Membrana Celular , Cricetinae , Cricetulus , Humanos , Camundongos , Transfecção , Água/metabolismo , Peixe-ZebraAssuntos
Anemia de Diamond-Blackfan/genética , Exoma , Haploinsuficiência , Mutação Puntual , RNA Ribossômico 18S/genética , Proteínas Ribossômicas/genética , Adulto , Processamento Alternativo , Anemia de Diamond-Blackfan/metabolismo , Anemia de Diamond-Blackfan/patologia , Animais , Sistemas CRISPR-Cas , Proliferação de Células , Pré-Escolar , Embrião não Mamífero , Éxons , Feminino , Edição de Genes , Expressão Gênica , Humanos , Lactente , Células K562 , Linhagem , RNA Ribossômico 18S/metabolismo , Proteínas Ribossômicas/deficiência , Irmãos , Sequenciamento do Exoma , Peixe-ZebraRESUMO
Diamond-Blackfan anaemia (DBA) is a cancer-prone genetic disorder characterized by pure red-cell aplasia and associated physical deformities. The ribosomal protein S19 gene (RPS19) is the most frequently mutated gene in DBA (~25%). TP53-mediated cell cycle arrest and/or apoptosis in erythroid cells have been suggested to be major factors for DBA development, but it is not clear why mutations in the ubiquitously expressed RPS19 gene specifically affect erythropoiesis. Previously, we showed that RPS19 deficiency in zebrafish recapitulates the erythropoietic and developmental phenotypes of DBA, including defective erythropoiesis with severe anaemia. In this study, we analysed the simultaneous loss-of-function of RPS19 and Tp53 in zebrafish to investigate the role of Tp53 in the erythroid and morphological defects associated with RPS19 deficiency. Co-inhibition of Tp53 activity rescued the morphological abnormalities, but did not alleviate erythroid aplasia in RPS19-deficient zebrafish. In addition, knockdown of two other RP genes, rps3a and rpl36a, which result in severe morphological abnormalities but only mild erythroid defects, also elicited an activated Tp53 response. These results suggest that a Tp53-independent but RPS19-dependent pathway could be responsible for defective erythropoiesis in RPS19-deficient zebrafish.
Assuntos
Anemia de Diamond-Blackfan/fisiopatologia , Eritropoese/fisiologia , Proteínas Ribossômicas/deficiência , Proteína Supressora de Tumor p53/fisiologia , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/metabolismo , Anemia de Diamond-Blackfan/patologia , Animais , Apoptose , Modelos Animais de Doenças , Genes p53 , RNA Mensageiro/genética , Proteínas Ribossômicas/genética , Proteína Supressora de Tumor p53/deficiência , Peixe-ZebraRESUMO
Ribosomes are responsible for protein synthesis in all cells. Ribosomal protein S19 (RPS19) is one of the 79 ribosomal proteins (RPs) in vertebrates. Heterozygous mutations in RPS19 have been identified in 25% of patients with Diamond-Blackfan anemia (DBA), but the relationship between RPS19 mutations and the pure red-cell aplasia of DBA is unclear. In this study, we developed an RPS19-deficient zebrafish by knocking down rps19 using a Morpholino antisense oligo. The RPS19-deficient animals showed a dramatic decrease in blood cells as well as deformities in the head and tail regions at early developmental stages. These phenotypes were rescued by injection of zebrafish rps19 mRNA, but not by injection of rps19 mRNAs with mutations that have been identified in DBA patients. Our results indicate that rps19 is essential for hematopoietic differentiation during early embryogenesis. The effects were specific to rps19, but knocking down the genes for three other RPs, rpl35, rpl35a and rplp2, produced similar phenotypes, suggesting that these genes might have a common function in zebrafish erythropoiesis. The RPS19-deficient zebrafish will provide a valuable tool for investigating the molecular mechanisms of DBA development in humans.
Assuntos
Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/embriologia , Proteínas Ribossômicas/deficiência , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/sangue , Peixe-Zebra/embriologia , Anemia de Diamond-Blackfan/genética , Animais , Sequência de Bases , Primers do DNA/genética , Modelos Animais de Doenças , Eritropoese/genética , Marcação de Genes , Humanos , Mutação , Fenótipo , RNA Antissenso/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Ribossômicas/sangue , Proteínas Ribossômicas/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/sangueRESUMO
OBJECTIVES: Cell-culture studies reported that prokaryotic RNA molecules among the various microbe-associated molecular patterns (MAMPs) were uniquely present in live bacteria and were categorized as viability-associated MAMPs. They also reported that specific nucleotide modifications are instrumental in the discrimination between self and nonself RNAs. The aim of this study was to characterize the in vivo immune induction potential of prokaryotic and eukaryotic ribosomal RNAs (rRNAs) using zebrafish embryos as novel whole animal model system. Additionally, we aimed to test the possible role of rRNA modifications in immune recognition. RESULTS: We used three immune markers to evaluate the induction potential of prokaryotic rRNA derived from Escherichia coli and eukaryotic rRNAs from chicken (nonself) and zebrafish (self). Lipopolysaccharide (LPS) of Pseudomonas aeruginosa served as a positive control. E. coli rRNA had an induction potential equivalent to that of LPS. The zebrafish innate immune system could discriminate between self and nonself rRNAs. Between the nonself rRNAs, E. coli rRNA was more immunogenic than chicken rRNA. The in vitro transcript of zebrafish 18S rRNA gene without the nucleotide modifications was not recognized by its own immune system. Our data suggested that prokaryotic rRNA is immunostimulatory in vivo and could be useful as an adjuvant.
Assuntos
Embrião não Mamífero/imunologia , Imunidade Inata , Células Procarióticas/metabolismo , RNA Ribossômico/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/imunologia , Animais , Biomarcadores/metabolismo , Lipopolissacarídeos/imunologia , RNA Ribossômico 18S/genética , Transcrição GênicaRESUMO
Many spliceosomal introns exist in the eukaryotic nuclear genome. Despite much research, the evolution of spliceosomal introns remains poorly understood. In this paper, we tried to gain insights into intron evolution from a novel perspective by comparing the gene structures of cytoplasmic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs), which are held to be of archaeal and bacterial origin, respectively. We analyzed 25 homologous pairs of CRP and MRP genes that together had a total of 527 intron positions. We found that all 12 of the intron positions shared by CRP and MRP genes resulted from parallel intron gains and none could be considered to be "conserved," i.e., descendants of the same ancestor. This was supported further by the high frequency of proto-splice sites at these shared positions; proto-splice sites are proposed to be sites for intron insertion. Although we could not definitively disprove that spliceosomal introns were already present in the last universal common ancestor, our results lend more support to the idea that introns were gained late. At least, our results show that MRP genes were intronless at the time of endosymbiosis. The parallel intron gains between CRP and MRP genes accounted for 2.3% of total intron positions, which should provide a reliable estimate for future inferences of intron evolution.
Assuntos
Evolução Molecular , Íntrons , Proteínas Ribossômicas/química , Sequência de Aminoácidos , Animais , Análise por Conglomerados , Éxons , Humanos , Modelos Genéticos , Modelos Estatísticos , Dados de Sequência Molecular , Proteínas Ribossômicas/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Spliceossomos/metabolismo , Fatores de TempoRESUMO
Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome characterized by red blood cell aplasia. Currently, mutations in 19 ribosomal protein genes have been identified in patients. However, the pathogenic mechanism of DBA remains unknown. Recently, several DBA models were generated in zebrafish (Danio rerio) to elucidate the molecular pathogenesis of disease and to explore novel treatments. Zebrafish have strong advantages in drug discovery due to their rapid development and transparency during embryogenesis and their applicability to chemical screens. Together with mice, zebrafish have now become a powerful tool for studying disease mechanisms and drug discovery. In this review, we introduce recent advances in DBA drug development and discuss the usefulness of zebrafish as a disease model.
RESUMO
Parkinson's disease (PD) is a neurodegenerative disease characterized by α-synuclein-positive inclusion bodies and loss of neurons, including dopaminergic neurons. Difficulty in replicating PD phenotypes using animal models partly limits the understanding of PD and the therapy required. Although PD is strongly associated with aging, most experimental animals may not exhibit age-related symptoms. Herein, we demonstrate that Nothobranchius furzeri, a rapidly aging teleost with a short life span, exhibits age-dependent degeneration of dopaminergic and noradrenergic neurons and progression of α-synuclein pathologies. These pathological phenotypes are similar to those observed in human patients with PD. Amelioration of the cell loss by genetic depletion of α-synuclein suggests that α-synuclein is not a bystander but a causative protein of neurodegeneration. N. furzeri can reveal mechanisms underlying PD, especially of the idiopathic form that affects a majority of patients with PD, including α-synuclein-dependent neurodegeneration, age-dependent phenotypes, and progression of α-synuclein pathology.
Assuntos
Dopamina/metabolismo , Neurônios/metabolismo , Doença de Parkinson/genética , alfa-Sinucleína/metabolismo , Animais , Modelos Animais de Doenças , Peixes , Fundulidae , HumanosRESUMO
Steroid sulfatase (STS) plays an important role in the regulation of steroid hormones. Metabolism of steroid hormones in zebrafish has been investigated, but the action of steroid sulfatase remains unknown. In this study, a zebrafish sts was cloned, expressed, purified, and characterized in comparison with the orthologous human enzyme. Enzymatic assays demonstrated that similar to human STS, zebrafish Sts was most active in catalyzing the hydrolysis of estrone-sulfate and estradiol-sulfate, among five steroid sulfates tested as substrates. Kinetic analyses revealed that the Km values of zebrafish Sts and human STS differed with respective substrates, but the catalytic efficiency as reflected by the Vmax/Km appeared comparable, except for DHEA-sulfate with which zebrafish Sts appeared less efficient. While zebrafish Sts was catalytically active at 28 °C, the enzyme appeared more active at 37 °C and with similar Km values to those determined at 28 °C. Assays performed in the presence of different divalent cations showed that the activities of both zebrafish and human STSs were stimulated by Ca2+, Mg2+, and Mn2+, and inhibited by Zn+2 and Fe2+. EMATE and STX64, two known mammalian steroid sulafatase inhibitors, were shown to be capable of inhibiting the activity of zebrafish Sts. Collectively, the results obtained indicated that zebrafish Sts exhibited enzymatic characteristics comparable to the human STS, suggesting that the physiological function of STS may be conserved between zebrafish and humans.