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1.
Elife ; 102021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34643182

RESUMO

The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. Here using Xenopus and mouse embryos, we establish molecular links between Tbx5 and retinoic acid (RA) signaling in the mesoderm and between RA signaling and sonic hedgehog expression in the endoderm to unveil a conserved RA-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary (CP) development. We demonstrate that Tbx5 directly maintains expression of aldh1a2, the RA-synthesizing enzyme, in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior. We find that Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the adjacent foregut endoderm through a conserved MACS1 enhancer. Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate expression of wnt2/2b, which induces pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing CP evolution and birth defects.


Assuntos
Família Aldeído Desidrogenase 1/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Coração/embriologia , Pulmão/embriologia , Retinal Desidrogenase/genética , Proteínas com Domínio T/genética , Proteínas de Xenopus/genética , Xenopus/embriologia , Família Aldeído Desidrogenase 1/metabolismo , Animais , Sequência de Bases , Mesoderma/embriologia , Camundongos , Retinal Desidrogenase/metabolismo , Alinhamento de Sequência , Proteínas com Domínio T/metabolismo , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo
2.
Cells ; 10(9)2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34572025

RESUMO

BACKGROUND: Most work in endocrinology focus on the action of a single hormone, and very little on the cross-talks between two hormones. Here we characterize the nature of interactions between thyroid hormone and glucocorticoid signaling during Xenopus tropicalis metamorphosis. METHODS: We used functional genomics to derive genome wide profiles of methylated DNA and measured changes of gene expression after hormonal treatments of a highly responsive tissue, tailfin. Clustering classified the data into four types of biological responses, and biological networks were modeled by system biology. RESULTS: We found that gene expression is mostly regulated by either T3 or CORT, or their additive effect when they both regulate the same genes. A small but non-negligible fraction of genes (12%) displayed non-trivial regulations indicative of complex interactions between the signaling pathways. Strikingly, DNA methylation changes display the opposite and are dominated by cross-talks. CONCLUSION: Cross-talks between thyroid hormones and glucocorticoids are more complex than initially envisioned and are not limited to the simple addition of their individual effects, a statement that can be summarized with the pseudo-equation: TH ∙ GC > TH + GC. DNA methylation changes are highly dynamic and buffered from genome expression.


Assuntos
Glucocorticoides/metabolismo , Metamorfose Biológica/fisiologia , Transdução de Sinais/fisiologia , Hormônios Tireóideos/metabolismo , Transcriptoma/genética , Xenopus/genética , Xenopus/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética
3.
Development ; 148(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34345915

RESUMO

The FET family of atypical RNA-binding proteins includes Fused in sarcoma (FUS), Ewing's sarcoma (EWS) and the TATA-binding protein-associate factor 15 (TAF15). FET proteins are highly conserved, suggesting specialized requirements for each protein. Fus regulates splicing of transcripts required for mesoderm differentiation and cell adhesion in Xenopus, but the roles of Ews and Taf15 remain unknown. Here, we analyze the roles of maternally deposited and zygotically transcribed Taf15, which is essential for the correct development of dorsoanterior neural tissues. By measuring changes in exon usage and transcript abundance from Taf15-depleted embryos, we found that Taf15 may regulate dorsoanterior neural development through fgfr4 and ventx2.1. Taf15 uses distinct mechanisms to downregulate Fgfr4 expression, namely retention of a single intron within fgfr4 when maternal and zygotic Taf15 is depleted, and reduction in the total fgfr4 transcript when zygotic Taf15 alone is depleted. The two mechanisms of gene regulation (post-transcriptional versus transcriptional) suggest that Taf15-mediated gene regulation is target and co-factor dependent, contingent on the milieu of factors that are present at different stages of development.


Assuntos
Encéfalo/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Xenopus/metabolismo , Animais , Encéfalo/fisiologia , Diferenciação Celular/fisiologia , Éxons/fisiologia , Feminino , Masculino , Neurônios/fisiologia , Xenopus/fisiologia
4.
DNA Repair (Amst) ; 106: 103193, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34339948

RESUMO

The nonhomologous DNA end joining pathway is required for repair of most double-strand breaks in the mammalian genome. Here we use a purified biochemical NHEJ system to compare the joining of free DNA with recombinant mononucleosomal and dinucleosomal substrates to investigate ligation and local DNA end resection. We find that the nucleosomal state permits ligation in a manner dependent on the presence of free DNA flanking the nucleosome core particle. Local resection at DNA ends by the Artemis:DNA-PKcs nuclease complex is completely suppressed in all mononucleosome substrates regardless of flanking DNA up to a length of 14 bp. Like mononucleosomes, dinucleosomes lacking flanking free DNA are not joined. Therefore, the nucleosomal state imposes severe constraints on NHEJ nuclease and ligase activities.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/metabolismo , Nucleossomos/metabolismo , Animais , Linhagem Celular , DNA/metabolismo , DNA Ligases/metabolismo , Células HeLa , Humanos , Spodoptera/metabolismo , Xenopus/metabolismo
5.
Nat Commun ; 12(1): 2802, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990563

RESUMO

Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET, patch-clamp fluorometry and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removing cAMP produced a largely rigid-body rotation of the C-linker relative to the transmembrane domain, bringing the A' helix of the C-linker in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not the presence of cAMP. These results suggest that - in contrast to electromechanical coupling for channel activation - the A' helix serves to couple the S4-helix movement for channel inactivation, which is likely a conserved mechanism for CNBD-family channels.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Animais , AMP Cíclico , Canais de Cátion Regulados por Nucleotídeos Cíclicos/antagonistas & inibidores , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Ativação do Canal Iônico , Masculino , Mecanotransdução Celular , Potenciais da Membrana , Modelos Moleculares , Oócitos/metabolismo , Técnicas de Patch-Clamp , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Ouriços-do-Mar/metabolismo , Espermatozoides/metabolismo , Xenopus/metabolismo
6.
Cells ; 10(3)2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33802526

RESUMO

Vertebrate postembryonic development is regulated by thyroid hormone (T3). Of particular interest is anuran metamorphosis, which offers several unique advantages for studying the role of T3 and its two nuclear receptor genes, TRα and TRß, during postembryonic development. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis animals and reported that TR is essential for the completion of metamorphosis. Furthermore, TRDKO tadpoles are stalled at the climax of metamorphosis before eventual death. Here we show that TRDKO intestine lacked larval epithelial cell death and adult stem cell formation/proliferation during natural metamorphosis. Interestingly, TRDKO tadpole intestine had premature formation of adult-like epithelial folds and muscle development. In addition, T3 treatment of premetamorphic TRDKO tadpoles failed to induce any metamorphic changes in the intestine. Furthermore, RNA-seq analysis revealed that TRDKO altered the expression of many genes in biological pathways such as Wnt signaling and the cell cycle that likely underlay the inhibition of larval epithelial cell death and adult stem cell development caused by removing both TR genes. Our data suggest that liganded TR is required for larval epithelial cell degeneration and adult stem cell formation, whereas unliganded TR prevents precocious adult tissue morphogenesis such as smooth-muscle development and epithelial folding.


Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Anfíbios/genética , Células Epiteliais/metabolismo , Intestinos/citologia , Larva/genética , Receptores dos Hormônios Tireóideos/genética , Hormônios Tireóideos/genética , Xenopus/genética , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Proteínas de Anfíbios/classificação , Proteínas de Anfíbios/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/genética , Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Ontologia Genética , Redes Reguladoras de Genes , Intestinos/efeitos dos fármacos , Intestinos/crescimento & desenvolvimento , Larva/citologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Redes e Vias Metabólicas/genética , Metamorfose Biológica , Anotação de Sequência Molecular , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Receptores dos Hormônios Tireóideos/deficiência , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/farmacologia , Via de Sinalização Wnt/genética , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismo
7.
J Med Chem ; 64(9): 5620-5631, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33902275

RESUMO

The α7 nicotinic acetylcholine receptor (nAChR) is present in the central nervous system and plays an important role in cognitive function and memory. α-Conotoxin LvIB, identified from genomic DNA of Conus lividus, its three isomers and four globular isomer analogues were synthesized and screened at a wide range of nAChR subtypes. One of the analogues, amidated [Q1G,ΔR14]LvIB, was found to be a potent blocker of rat α7 nAChRs. Importantly, it differentiates between α7 nAChRs of human (IC50: 1570 nM) and rat (IC50: 97 nM). Substitutions between rat and human α7 nAChRs at three key mutation sites revealed that no single mutant could completely change the activity profile of amidated [Q1G,ΔR14]LvIB. Rather, we found that the combined influence of Gln141, Asn184, and Lys186 determines the α7 nAChR species specificity of this peptide. This engineered α4/4 conotoxin has potential applications as a template for designing ligands to selectively block human α7 nAChRs.


Assuntos
Conotoxinas/química , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Conotoxinas/síntese química , Conotoxinas/metabolismo , Humanos , Concentração Inibidora 50 , Isomerismo , Ligantes , Simulação de Dinâmica Molecular , Mutagênese , Oócitos/metabolismo , Ratos , Alinhamento de Sequência , Especificidade da Espécie , Xenopus/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Receptor Nicotínico de Acetilcolina alfa7/genética
8.
J Med Chem ; 64(8): 5185-5197, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33851832

RESUMO

Bombinins are a wide family of antimicrobial peptides from Xenopus skin. By sequence clustering, we highlighted at least three families named A, B, and H, which might exert antibacterial activity by different modes of action. In this work, we study bombinin-like peptide 3 (BLP-3) as a nonhemolytic representative of the quite unexplored class A due to its appealing activity toward WHO-priority-list bacteria such as Neisseria, Pseudomonas aeruginosa, and Staphylococcus aureus. A marked preference for cardiolipin and phosphatidylglycerol head groups, typically found in bacteria, is proven with biomimetic membranes studied by liquid and solid NMR and MD simulations. BLP-3 gets structured upon interaction and penetrates deeply into the bilayer in two steps involving a superficial insertion of key side chains and subsequent internalization. All along the pathway, a fundamental role is played by lysine residues in the conserved region 11-19, which act in synergy with other key residues.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Materiais Biomiméticos/metabolismo , Bicamadas Lipídicas/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/classificação , Materiais Biomiméticos/química , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Pele/metabolismo , Xenopus/metabolismo
9.
J Cell Physiol ; 236(9): 6344-6361, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33521982

RESUMO

Melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R), two neural G protein-coupled receptors are known to be functionally critical for energy balance in vertebrates. As allosteric regulators of melanocortin receptors, melanocortin accessory proteins (MRAPs) are also involved in energy homeostasis. The interaction of MRAPs and melanocortin signaling was previously shown in mammals and zebrafish, but nothing had been reported in amphibians. As the basal class of tetrapods, amphibians occupy a phylogenetic transition between teleosts and terrestrial animals. Here we examined the evolutionary conservation of MC3R, MC4R, and MRAPs between diploid Xenopus tropicalis (xt-) and other chordates and investigated the pharmacological regulatory properties of MRAPs on the neural MC3R and MC4R signaling. Our results showed that xtMRAP and xtMRAP2 both exerted robust potentiation effect on agonist (α-MSH and adrenocorticotropin [ACTH]) induced activation and modulated the basal activity and cell surface translocation of xtMC3R and xtMC4R. In addition, the presence of two accessory proteins could convert xtMC3R and xtMC4R into ACTH-preferred receptors. These findings suggest that the presence of MRAPs exhibits fine control over the pharmacological activities of the neuronal MC3R and MC4R signaling in the Xenopus tropicalis, which is physiologically relevant with the complicated transition of feeding behaviors during their life history.


Assuntos
Melanocortinas/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Sequência de Aminoácidos , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cromossomos/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Filogenia , Ligação Proteica/efeitos dos fármacos , Receptores de Melanocortina/química , Receptores de Melanocortina/metabolismo , Sintenia/genética , Distribuição Tecidual , Xenopus/genética , Proteínas de Xenopus/química , alfa-MSH/farmacologia
10.
Dev Biol ; 473: 59-70, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33484704

RESUMO

Xenopus tadpoles are a unique model for regeneration in that they exhibit two distinct phases of age-specific regenerative competence. In Xenopus laevis, young tadpoles fully regenerate following major injuries such as tail transection, then transiently lose regenerative competence during the "refractory period" from stages 45-47. Regenerative competence is then regained in older tadpoles before being permanently lost during metamorphosis. Here we show that a similar refractory period exists in X. tropicalis. Notably, tadpoles lose regenerative competence gradually in X. tropicalis, with full regenerative competence lost at stage 47. We find that the refractory period coincides closely with depletion of maternal yolk stores and the onset of independent feeding, and so we hypothesized that it might be caused in part by nutrient stress. In support of this hypothesis, we find that cell proliferation declines throughout the tail as the refractory period approaches. When we block nutrient mobilization by inhibiting mTOR signaling, we find that tadpole growth and regeneration are reduced, while yolk stores persist. Finally, we are able to restore regenerative competence and cell proliferation during the refractory period by abundantly feeding tadpoles. Our study argues that nutrient stress contributes to lack of regenerative competence and introduces the X. tropicalis refractory period as a valuable new model for interrogating how metabolic constraints inform regeneration.


Assuntos
Regeneração/fisiologia , Cauda/fisiologia , Xenopus/embriologia , Animais , Proliferação de Células , Gema de Ovo , Larva/metabolismo , Metamorfose Biológica/fisiologia , Nutrientes , Transdução de Sinais , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo
11.
Nat Commun ; 12(1): 612, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504787

RESUMO

The motile cilia of ependymal cells coordinate their beats to facilitate a forceful and directed flow of cerebrospinal fluid (CSF). Each cilium originates from a basal body with a basal foot protruding from one side. A uniform alignment of these basal feet is crucial for the coordination of ciliary beating. The process by which the basal foot originates from subdistal appendages of the basal body, however, is unresolved. Here, we show FGFR1 Oncogene Partner (FOP) is a useful marker for delineating the transformation of a circular, unpolarized subdistal appendage into a polarized structure with a basal foot. Ankyrin repeat and SAM domain-containing protein 1A (ANKS1A) interacts with FOP to assemble region I of the basal foot. Importantly, disruption of ANKS1A reduces the size of region I. This produces an unstable basal foot, which disrupts rotational polarity and the coordinated beating of cilia in young adult mice. ANKS1A deficiency also leads to severe degeneration of the basal foot in aged mice and the detachment of cilia from their basal bodies. This role of ANKS1A in the polarization of the basal foot is evolutionarily conserved in vertebrates. Thus, ANKS1A regulates FOP to build and maintain the polarity of subdistal appendages.


Assuntos
Cílios/metabolismo , Simulação de Dinâmica Molecular , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Envelhecimento/patologia , Animais , Corpos Basais/metabolismo , Evolução Biológica , Cílios/ultraestrutura , Embrião não Mamífero/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Fatores de Transcrição/metabolismo , Xenopus/embriologia , Xenopus/metabolismo
12.
Thyroid ; 31(4): 692-702, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33076783

RESUMO

Background: Thyroid hormone (triiodothyronine [T3]) plays an important role in regulating vertebrate developmental, cellular, and metabolic processes via T3 receptor (TR). Liganded TR recruit coactivator complexes that include steroid receptor coactivators (SRC1, SRC2 or SRC3), which are histone acetyltransferases, to T3-responsive promoters. The functions of endogenous coactivators during T3-dependent mammalian adult organ development remain largely unclear, in part, due to the difficulty to access and manipulate late-stage embryos and neonates. We use Xenopus metamorphosis as a model for postembryonic development in vertebrates. This process is controlled by T3, involves drastic changes in every organ/tissue, and can be easily manipulated. We have previously found that SRC3 was upregulated in the intestine during amphibian metamorphosis. Methods: To determine the function of endogenous SRC3 during intestinal remodeling, we have generated Xenopus tropicalis animals lacking a functional SRC3 gene and analyzed the resulting phenotype. Results: Although removing SRC3 had no apparent effect on external development and animal gross morphology, the SRC3 (-/-) tadpoles displayed a reduction in the acetylation of histone H4 in the intestine compared with that in wild-type animals. Further, the expression of TR target genes was also reduced in SRC3 (-/-) tadpoles during intestinal remodeling. Importantly, SRC3 (-/-) tadpoles had inhibited/delayed intestinal remodeling during natural and T3-induced metamorphosis, including reduced adult intestinal stem cell proliferation and apoptosis of larval epithelial cells. Conclusion: Our results, thus, demonstrate that SRC3 is a critical component of the TR-signaling pathway in vivo during intestinal remodeling.


Assuntos
Intestinos/crescimento & desenvolvimento , Metamorfose Biológica , Coativador 3 de Receptor Nuclear/metabolismo , Células-Tronco/metabolismo , Tri-Iodotironina/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Animais , Apoptose , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Coativador 3 de Receptor Nuclear/genética , Transdução de Sinais , Xenopus/genética , Xenopus/crescimento & desenvolvimento , Proteínas de Xenopus/genética
13.
Int J Biol Macromol ; 167: 719-725, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33279564

RESUMO

Interferon regulatory factor 1 (IRF1) is an important regulator in controlling the transcription of type I interferon genes, and its functions have been well-characterized in mammals, birds and fish. However, little information is available regarding the function of amphibian IRF1. In this study, an IRF1 gene homolog named as Xt-IRF1 was identified in the Western clawed frog (Xenopus tropicalis), an amphibian model specie widely used for comparative immunology research. Xt-IRF1 and IRF1 in other vertebrates possess similar genomic structure and flanking genes, and were grouped together to form a separate clade in phylogenetic tree. In addition, Xt-IRF1 gene was constitutively expressed in all tissues examined, with the highest expression level observed in spleen, and was inducible after poly(I:C) stimulation. Importantly, the expression of Xt-IRF1 was markedly induced by recombinant type I interferon, and Xt-IRF1 induced a strong activation of both IFNß and ISRE promoters. The present study opens the door to investigate the roles of IRF1 in amphibians, and thus contributes to a better understanding of the functional evolution of IRFs in lower tetrapods.


Assuntos
Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Xenopus/genética , Anfíbios/genética , Anfíbios/metabolismo , Animais , Clonagem Molecular , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Genoma , Genômica , Fator Regulador 1 de Interferon/química , Filogenia , Regiões Promotoras Genéticas , Elementos de Resposta , Análise de Sequência , Xenopus/classificação , Xenopus/metabolismo
14.
Nucleic Acids Res ; 49(D1): D254-D260, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33035346

RESUMO

Transfer RNA-derived fragments (tRFs) are a new class of small non-coding RNAs and play important roles in biological and physiological processes. Prediction of tRF target genes and binding sites is crucial in understanding the biological functions of tRFs in the molecular mechanisms of human diseases. We developed a publicly accessible web-based database, tRFtarget (http://trftarget.net), for tRF target prediction. It contains the computationally predicted interactions between tRFs and mRNA transcripts using the two state-of-the-art prediction tools RNAhybrid and IntaRNA, including location of the binding sites on the target, the binding region, and free energy of the binding stability with graphic illustration. tRFtarget covers 936 tRFs and 135 thousand predicted targets in eight species. It allows researchers to search either target genes by tRF IDs or tRFs by gene symbols/transcript names. We also integrated the manually curated experimental evidence of the predicted interactions into the database. Furthermore, we provided a convenient link to the DAVID® web server to perform downstream functional pathway analysis and gene ontology annotation on the predicted target genes. This database provides useful information for the scientific community to experimentally validate tRF target genes and facilitate the investigation of the molecular functions and mechanisms of tRFs.


Assuntos
Bases de Dados de Ácidos Nucleicos , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , RNA de Transferência/genética , Animais , Pareamento de Bases , Sequência de Bases , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ontologia Genética , Humanos , Camundongos , Anotação de Sequência Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Termodinâmica , Xenopus/genética , Xenopus/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
15.
Development ; 148(2)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33318149

RESUMO

Mutations in the RNA helicase DDX3 have emerged as a frequent cause of intellectual disability in humans. Because many individuals carrying DDX3 mutations have additional defects in craniofacial structures and other tissues containing neural crest (NC)-derived cells, we hypothesized that DDX3 is also important for NC development. Using Xenopus tropicalis as a model, we show that DDX3 is required for normal NC induction and craniofacial morphogenesis by regulating AKT kinase activity. Depletion of DDX3 decreases AKT activity and AKT-dependent inhibitory phosphorylation of GSK3ß, leading to reduced levels of ß-catenin and Snai1: two GSK3ß substrates that are crucial for NC induction. DDX3 function in regulating these downstream signaling events during NC induction is likely mediated by RAC1, a small GTPase whose translation depends on the RNA helicase activity of DDX3. These results suggest an evolutionarily conserved role of DDX3 in NC development by promoting AKT activity, and provide a potential mechanism for the NC-related birth defects displayed by individuals harboring mutations in DDX3 and its downstream effectors in this signaling cascade.


Assuntos
RNA Helicases DEAD-box/metabolismo , Crista Neural/embriologia , Crista Neural/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Xenopus/metabolismo , Animais , Cartilagem/embriologia , Cartilagem/metabolismo , Embrião não Mamífero/metabolismo , Face/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Glicogênio Sintase Quinase 3 beta/metabolismo , Morfogênese/genética , Fosforilação , Estabilidade Proteica , Crânio/embriologia , Crânio/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Via de Sinalização Wnt , Xenopus/genética , beta Catenina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
16.
Thyroid ; 31(1): 128-142, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32515287

RESUMO

Background: There are two highly conserved thyroid hormone (triiodothyronine [T3]) receptor (TR) genes, TRα and TRß, in all vertebrates, and the expression of TRα but not TRß is activated earlier than T3 synthesis during development. In human, high levels of T3 are present during the several months around birth, and T3 deficiency during this period causes severe developmental abnormalities including skeletal and intestinal defects. It is, however, difficult to study this period in mammals as the embryos and neonates depend on maternal supply of nutrients for survival. However, Xenopus tropicalis undergoes a T3-dependent metamorphosis, which drastically changes essentially every organ in a tadpole. Of interest is intestinal remodeling, which involves near complete degeneration of the larval epithelium through apoptosis. Concurrently, adult intestinal stem cells are formed de novo and subsequently give rise to the self-renewing adult epithelial system, resembling intestinal maturation around birth in mammals. We have previously demonstrated that T3 signaling is essential for the formation of adult intestinal stem cells during metamorphosis. Methods: We studied the function of endogenous TRα in the tadpole intestine by using knockout animals and RNA-seq analysis. Results: We observed that removing endogenous TRα caused defects in intestinal remodeling, including drastically reduced larval epithelial cell death and adult intestinal stem cell proliferation. Using RNA-seq on intestinal RNA from premetamorphic wild-type and TRα-knockout tadpoles treated with or without T3 for one day, before any detectable T3-induced cell death and stem cell formation in the tadpole intestine, we identified more than 1500 genes, which were regulated by T3 treatment of the wild-type but not TRα-knockout tadpoles. Gene Ontology and biological pathway analyses revealed that surprisingly, these TRα-regulated genes were highly enriched with cell cycle-related genes, in addition to genes related to stem cells and apoptosis. Conclusions: Our findings suggest that TRα-mediated T3 activation of the cell cycle program is involved in larval epithelial cell death and adult epithelial stem cell development during intestinal remodeling.


Assuntos
Células-Tronco Adultas/metabolismo , Ciclo Celular , Proliferação de Células , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Receptores alfa dos Hormônios Tireóideos/deficiência , Tri-Iodotironina/metabolismo , Proteínas de Xenopus/deficiência , Xenopus/metabolismo , Células-Tronco Adultas/patologia , Animais , Apoptose , Células Epiteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento , Mucosa Intestinal/patologia , Larva/genética , Larva/metabolismo , Metamorfose Biológica , Transdução de Sinais , Receptores alfa dos Hormônios Tireóideos/genética , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus/genética
17.
Cell Mol Life Sci ; 78(3): 1051-1064, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32472188

RESUMO

Nicotinic acetylcholine receptors (nAChRs) are pentameric ion channels expressed in the central nervous systems. nAChRs containing the α4, ß2 and α5 subunits are specifically involved in addictive processes, but their functional architecture is poorly understood due to the intricacy of assembly of these subunits. Here we constrained the subunit assembly by designing fully concatenated human α4ß2 and α4ß2α5 receptors and characterized their properties by two-electrodes voltage-clamp electrophysiology in Xenopus oocytes. We found that α5-containing nAChRs are irreversibly blocked by methanethiosulfonate (MTS) reagents through a covalent reaction with a cysteine present only in α5. MTS-block experiments establish that the concatemers are expressed in intact form at the oocyte surface, but that reconstitution of nAChRs from loose subunits show inefficient and highly variable assembly of α5 with α4 and ß2. Mutational analysis shows that the concatemers assemble both in clockwise and anticlockwise orientations, and that α5 does not contribute to ACh binding from its principal (+) site. Reinvestigation of suspected α5-ligands such as galantamine show no specific effect on α5-containing concatemers. Analysis of the α5-D398N mutation that is linked to smoking and lung cancer shows no significant effect on the electrophysiological function, suggesting that its effect might arise from alteration of other cellular processes. The concatemeric strategy provides a well-characterized platform for mechanistic analysis and screening of human α5-specific ligands.


Assuntos
Receptores Nicotínicos/metabolismo , Regiões 5' não Traduzidas , Acetilcolina/química , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Mesilatos/farmacologia , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oócitos/fisiologia , Oxidiazóis/farmacologia , Técnicas de Patch-Clamp , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Piridinas/farmacologia , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismo , Proteínas de Xenopus/genética , Globinas beta/genética
18.
Genes (Basel) ; 11(11)2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33218193

RESUMO

Gastrulation drives the establishment of three germ layers and embryonic axes during frog embryonic development. Mesodermal cell fate specification and morphogenetic movements are vital factors coordinating gastrulation, which are regulated by numerous signaling pathways, such as the Wnt (Wingless/Integrated), Notch, and FGF (Fibroblast growth factor) pathways. However, the coordination of the Notch and FGF signaling pathways during gastrulation remains unclear. We identified a novel helix-loop-helix DNA binding domain gene (Hes5.9), which was regulated by the FGF and Notch signaling pathways during gastrulation. Furthermore, gain- and loss-of-function of Hes5.9 led to defective cell migration and disturbed the expression patterns of mesodermal and endodermal marker genes, thus interfering with gastrulation. Collectively, these results suggest that Hes5.9 plays a crucial role in cell fate decisions and cell migration during gastrulation, which is modulated by the FGF and Notch signaling pathways.


Assuntos
Embrião não Mamífero/citologia , Fatores de Crescimento de Fibroblastos/metabolismo , Receptores Notch/metabolismo , Proteínas de Xenopus/genética , Xenopus/embriologia , Animais , Diferenciação Celular , Movimento Celular , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Endoderma , Feminino , Gastrulação/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma , Microinjeções , Pirróis/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo
19.
DNA Repair (Amst) ; 96: 102973, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32987353

RESUMO

TOPBP1 is an important scaffold protein that helps orchestrate the cellular response to DNA damage. Although it has been previously appreciated that TOPBP1 can form oligomers, how this occurs and the functional consequences for oligomerization were not yet known. Here, we use protein binding assays and other biochemical techniques to study how TOPBP1 self associates. TOPBP1 contains 9 copies of the BRCT domain, and we report that a subset of these BRCT domains interact with one another to drive oligomerization. An intact BRCT 2 domain is required for TOPBP1 oligomerization and we find that the BRCT1&2 region of TOPBP1 interacts with itself and with the BRCT4&5 pair. RAD9 and RHINO are two heterologous binding partners for TOPBP1's BRCT 1&2 domains, and we show that binding of these partners does not come at the expense of TOPBP1 oligomerization. Furthermore, we show that a TOPBP1 oligomer can simultaneously interact with both RAD9 and RHINO. Lastly, we find that the oligomeric state necessary for TOPBP1 to activate the ATR protein kinase is likely to be a tetramer.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transdução de Sinais , Xenopus/genética , Xenopus/metabolismo
20.
Nature ; 585(7826): 563-568, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939088

RESUMO

Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7-10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR-Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.


Assuntos
Endotelinas/metabolismo , Evolução Molecular , Crista Neural/citologia , Petromyzon/metabolismo , Transdução de Sinais , Xenopus/metabolismo , Animais , Desenvolvimento Ósseo , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Linhagem da Célula , Endotelinas/genética , Feminino , Cabeça/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Ligantes , Masculino , Petromyzon/genética , Petromyzon/crescimento & desenvolvimento , Receptores de Endotelina/deficiência , Receptores de Endotelina/genética , Receptores de Endotelina/metabolismo , Xenopus/genética , Xenopus/crescimento & desenvolvimento
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