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1.
Cell ; 178(1): 107-121.e18, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31251911

RESUMO

Increasing evidence suggests that transcriptional control and chromatin activities at large involve regulatory RNAs, which likely enlist specific RNA-binding proteins (RBPs). Although multiple RBPs have been implicated in transcription control, it has remained unclear how extensively RBPs directly act on chromatin. We embarked on a large-scale RBP ChIP-seq analysis, revealing widespread RBP presence in active chromatin regions in the human genome. Like transcription factors (TFs), RBPs also show strong preference for hotspots in the genome, particularly gene promoters, where their association is frequently linked to transcriptional output. Unsupervised clustering reveals extensive co-association between TFs and RBPs, as exemplified by YY1, a known RNA-dependent TF, and RBM25, an RBP involved in splicing regulation. Remarkably, RBM25 depletion attenuates all YY1-dependent activities, including chromatin binding, DNA looping, and transcription. We propose that various RBPs may enhance network interaction through harnessing regulatory RNAs to control transcription.


Assuntos
Cromatina/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Transcrição Gênica/genética , Fator de Transcrição YY1/metabolismo , Sítios de Ligação , Regulação da Expressão Gênica , Genoma Humano/genética , Células Hep G2 , Humanos , Células K562 , Proteínas Nucleares , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas de Ligação a RNA/genética , RNA-Seq , Transcriptoma , Fator de Transcrição YY1/genética
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.
Mol Cell ; 68(4): 745-757.e5, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29104020

RESUMO

R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed.


Assuntos
DNA/química , Ácidos Nucleicos Heteroduplexes/química , Regiões Promotoras Genéticas/fisiologia , RNA/química , Ribonuclease H/química , Transcrição Gênica , DNA/biossíntese , Células HEK293 , Humanos , Células K562 , Ácidos Nucleicos Heteroduplexes/metabolismo , RNA/biossíntese
4.
PLoS Genet ; 18(3): e1009841, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35245286

RESUMO

Neural retina leucine zipper (NRL) is an essential gene for the fate determination and differentiation of the precursor cells into rod photoreceptors in mammals. Mutations in NRL are associated with the autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in the zebrafish (Danio rerio), a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model via the CRISPR-Cas9 technology and observed a surprising phenotype characterized by a reduced number, but not the total loss, of rods and over-growth of green cones. We discovered two waves of rod genesis, nrl-dependent and -independent at the embryonic and post-embryonic stages, respectively, in zebrafish by monitoring the rod development. Through bulk and single-cell RNA sequencing, we characterized the gene expression profiles of the whole retina and each retinal cell type from the wild type and nrl knockout zebrafish. The over-growth of green cones and mis-expression of green-cone-specific genes in rods in nrl mutants suggested that there are rod/green-cone bipotent precursors, whose fate choice between rod versus green-cone is controlled by nrl. Besides, we identified the mafba gene as a novel regulator of the nrl-independent rod development, based on the cell-type-specific expression patterns and the retinal phenotype of nrl/mafba double-knockout zebrafish. Gene collinearity analysis revealed the evolutionary origin of mafba and suggested that the function of mafba in rod development is specific to modern fishes. Furthermore, the altered photoreceptor composition and abnormal gene expression in nrl mutants caused progressive retinal degeneration and subsequent regeneration. Accordingly, this study revealed a novel function of the mafba gene in rod development and established a working model for the developmental and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish.


Assuntos
Degeneração Retiniana , Peixe-Zebra , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas do Olho/metabolismo , Mamíferos/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
5.
EMBO Rep ; 23(6): e54387, 2022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35532311

RESUMO

Primordial germ cells (PGCs) are the progenitor cells that give rise to sperm and eggs. Sinhcaf is a recently identified subunit of the Sin3 histone deacetylase complex (SIN3A-HDAC). Here, we provide evidence that Sinhcaf-dependent histone deacetylation is essential for germ plasm aggregation and primordial germ cell specification. Specifically, maternal-zygotic sinhcaf zebrafish mutants exhibit germ plasm aggregation defects, decreased PGC abundance and male-biased sex ratio, which can be rescued by re-expressing sinhcaf. Overexpression of sinhcaf results in excess PGCs and a female-biased sex ratio. Sinhcaf binds to the promoter region of kif26ab. Loss of sinhcaf epigenetically switches off kif26ab expression by increasing histone 3 acetylation in the promoter region. Injection of kif26ab mRNA could partially rescue the germ plasm aggregation defects in sinhcaf mutant embryos. Taken together, we demonstrate a role of Sinhcaf in germ plasm aggregation and PGC specialization that is mediated by regulating the histone acetylation status of the kif26ab promoter to activate its transcription. Our findings provide novel insights into the function and regulatory mechanisms of Sinhcaf-mediated histone deacetylation in PGC specification.


Assuntos
Histonas , Peixe-Zebra , Animais , Feminino , Células Germinativas/metabolismo , Histonas/metabolismo , Masculino , RNA Mensageiro/genética , Peixe-Zebra/genética , Zigoto
6.
Fish Shellfish Immunol ; 154: 109979, 2024 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-39442737

RESUMO

Ichthyophthiriasis (Ich), also known as white spot disease, causes significant economic losses to fish farmers once an outbreak occurs. For fish survival, it is therefore crucial to understand the pathogenic mechanism and find effective prevention methods. In this study, we obtained data for four stages (theront, trophont, protomont and tomont) of Ichthyophthirius multifiliis by single-cell RNA sequencing (scRNA-seq). We found that the invasion-related proteins encoded by highly expressed genes in the theront stage mainly belong to the leishmanolysin family proteins, heat shock proteins, transmembrane proteins and cysteine proteases (CPs). Additionally, the exosome pathway appears to play a significant role in the invasion process of the theront. Since cysteine proteases are expressed at all stages of the I. multifiliis, and five CP-related genes were significantly upregulated at the theront stage of its life cycle - two of which are enriched in the exosome pathway - we incubated I. multifiliis theronts and protomonts with cysteine protease inhibitor (E-64). Our findings revealed that E-64 could kill both stages of the parasite in vitro and affected tomont division and subsequent release. Furthermore, infection experiment showed that E-64 could significantly inhibit the invasion of theronts. Based on our preliminary analysis from the transcriptomic and E-64 experiments, we have confirmed that CPs play a crucial role in I. multifiliis. This research establishes a foundation for future strategies in the prevention and control of Ich.

7.
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
8.
Blood ; 133(8): 805-815, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30482793

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) originate from the hemogenic endothelium via the endothelial-to-hematopoietic transition, are self-renewing, and replenish all lineages of blood cells throughout life. BCAS2 (breast carcinoma amplified sequence 2) is a component of the spliceosome and is involved in multiple biological processes. However, its role in hematopoiesis remains unknown. We established a bcas2 knockout zebrafish model by using transcription activator-like effector nucleases. The bcas2 -/- zebrafish showed severe impairment of HSPCs and their derivatives during definitive hematopoiesis. We also observed significant signs of HSPC apoptosis in the caudal hematopoietic tissue of bcas2 -/- zebrafish, which may be rescued by suppression of p53. Furthermore, we show that the bcas2 deletion induces an abnormal alternative splicing of Mdm4 that predisposes cells to undergo p53-mediated apoptosis, which provides a mechanistic explanation of the deficiency observed in HSPCs. Our findings revealed a novel and vital role for BCAS2 during HSPC maintenance in zebrafish.


Assuntos
Embrião não Mamífero/embriologia , Desenvolvimento Embrionário , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Técnicas de Silenciamento de Genes , Proteínas de Neoplasias/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
9.
Hum Genet ; 137(10): 779-794, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30242501

RESUMO

Most cases of Usher syndrome type II (USH2) are due to mutations in the USH2A gene. There are no effective treatments or ideal animal models for this disease, and the pathological mechanisms of USH2 caused by USH2A mutations are still unknown. Here, we constructed a ush2a knockout (ush2a-/-) zebrafish model using TALEN technology to investigate the molecular pathology of USH2. An early onset auditory disorder and abnormal morphology of inner ear stereocilia were identified in the ush2a-/- zebrafish. Consequently, the disruption of Ush2a in zebrafish led to a hearing impairment, like that in mammals. Electroretinography (ERG) test indicated that deletion of Ush2a affected visual function at an early stage, and histological analysis revealed that the photoreceptors progressively degenerated. Rod degeneration occurred prior to cone degeneration in ush2a-/- zebrafish, which is consistent with the classical description of the progression of retinitis pigmentosa (RP). Destruction of the outer segments (OSs) of rods led to the down-regulation of phototransduction cascade proteins at late stage. The expression of Ush1b and Ush1c was up-regulated when Ush2a was null. We also found that disruption of fibronectin assembly at the retinal basement membrane weakened cell adhesion in ush2a-/- mutants. In summary, for the first time, we generated a ush2a knockout zebrafish line with auditory disorder and retinal degeneration which mimicked the symptoms of patients, and revealed that disruption of fibronectin assembly may be one of the factors underlying RP. This model may help us to better understand the pathogenic mechanism and find treatment for USH2 in the future.


Assuntos
Proteínas da Matriz Extracelular , Técnicas de Inativação de Genes , Síndromes de Usher , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Modelos Animais de Doenças , Eletrorretinografia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Humanos , Síndromes de Usher/genética , Síndromes de Usher/metabolismo , Síndromes de Usher/patologia , Síndromes de Usher/fisiopatologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
10.
Methods ; 69(1): 58-66, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24556556

RESUMO

Transcription activator like effector nucleases (TALENs) is a promising approach to disrupt intended genomic loci. The assembly of highly effective TALENs is critical for successful genome editing. Recently we reported a convenient and robust platform to construct customized TALENs. The TALENs generated by this platform have been proven to be highly effective for gene disruption in Xenopus tropicalis and zebrafish as well as large genomic deletions in zebrafish. The one-time success rate of targeted gene disruption is about 90% for more than 100 genomic loci tested, with the mutation frequencies often reaching above 50%. Here we describe the validated protocol for TALEN assembly, methods for generating gene knockout animals in X. tropicalis and zebrafish, as well as the protocol for engineering large genomic deletions in zebrafish.


Assuntos
Técnicas de Inativação de Genes , Xenopus/genética , Peixe-Zebra/genética , Animais , Desoxirribonucleases/genética , Deleção de Genes , Engenharia Genética/métodos , Genoma , Microinjeções , Xenopus/embriologia , Peixe-Zebra/embriologia
11.
Ultrastruct Pathol ; 39(5): 324-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26107819

RESUMO

This study aimed at analyzing the cytotoxicity and pathological effects of cadmium on the ovary. Our studies revealed that cadmium was deposited in the mouse ovary after 8 d cadmium injection in vivo. Also, the increase in the rate of body weight was slowed, while the gonadosomatic index was reduced in the CdCl2 group, compared with the control group. Meanwhile, cadmium affected the maturation of follicles, the degradation of corpus luteum, the arrangement of follicles and corpus luteum, and increased the number of atresia follicles. Besides, under the electron microscope, chromatin margination, karopyknosis, swelling of mature cisternae of Golgi apparatus, mitochondrial cristae disappearance, and swelling of the rough endoplasmic reticulum can be observed in the CdCl2 group mice. Collectively, our findings elucidated the morphological mechanism that the exposure of cadmium changed the ultrastructure of cells in ovary tissues.


Assuntos
Cádmio/toxicidade , Ovário/efeitos dos fármacos , Ovário/patologia , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Camundongos , Microscopia Eletrônica de Transmissão , Espectrofotometria Atômica
12.
Sci Data ; 11(1): 675, 2024 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-38909036

RESUMO

The greater amberjack is a very important fishery species with high commercial value, and it is distributed worldwide. Transcriptome-based studies on S. dumerili have been limited by an inadequate reference genome and a lack of well-annotated full-length transcripts. In this study, a total of 12 tissues from juvenile and adult fish both sexes were collected for next-generation RNA sequencing (RNA-seq) and full-length isoform sequencing (Iso-seq). For Iso-seq, a total of 163,218, 149,716, and 189,169 high-quality unique transcript sequences were obtained, with an N50 of 5,441, 5,255, and 5,939, from juvenile, adult male and adult female S. dumerili, respectively. We integrated the Iso-seq and RNA-seq data to construct a comprehensive gene annotation and systematically profiled the dynamics of gene expression across the 12 tissues. Our gene models had greater detail and accuracy than those from NCBI and Ensembl, with more precise polyA locations. These resources serve as a foundation for functional genomic studies and provide valuable insights into the molecular mechanisms underlying the development, reproduction and commercial traits of amberjack.


Assuntos
Genoma , RNA-Seq , Animais , Masculino , Feminino , Transcriptoma , Anotação de Sequência Molecular , Análise de Sequência de RNA , Sequenciamento de Nucleotídeos em Larga Escala , Peixes/genética
13.
Front Genet ; 15: 1409016, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39055259

RESUMO

Introduction: Inherited retinal diseases (IRDs) affect ∼4.5 million people worldwide. Elusive pathogenic variants in over 280 genes are associated with one or more clinical forms of IRDs. It is necessary to understand the complex interaction among retinal cell types and pathogenic genes by constructing a regulatory network. In this study, we attempt to establish a panoramic expression view of the cooperative work in retinal cells to understand the clinical manifestations and pathogenic bases underlying IRDs. Methods: Single-cell RNA sequencing (scRNA-seq) data on the retinas from 35 retina samples of 3 species (human, mouse, and zebrafish) including 259,087 cells were adopted to perform a comparative analysis across species. Bioinformatic tools were used to conduct weighted gene co-expression network analysis (WGCNA), single-cell regulatory network analysis, cell-cell communication analysis, and trajectory inference analysis. Results: The cross-species comparison revealed shared or species-specific gene expression patterns at single-cell resolution, such as the stathmin family genes, which were highly expressed specifically in zebrafish Müller glias (MGs). Thirteen gene modules were identified, of which nine were associated with retinal cell types, and Gene Ontology (GO) enrichment of module genes was consistent with cell-specific highly expressed genes. Many IRD genes were identified as hub genes and cell-specific regulons. Most IRDs, especially the retinitis pigmentosa (RP) genes, were enriched in rod-specific regulons. Integrated expression and transcription regulatory network genes, such as congenital stationary night blindness (CSNB) genes GRK1, PDE6B, and TRPM1, showed cell-specific expression and transcription characteristics in either rods or bipolar cells (BCs). IRD genes showed evolutionary conservation (GNAT2, PDE6G, and SAG) and divergence (GNAT2, MT-ND4, and PDE6A) along the trajectory of photoreceptors (PRs) among species. In particular, the Leber congenital amaurosis (LCA) gene OTX2 showed high expression at the beginning of the trajectory of both PRs and BCs. Conclusion: We identified molecular pathways and cell types closely connected with IRDs, bridging the gap between gene expression, genetics, and pathogenesis. The IRD genes enriched in cell-specific modules and regulons suggest that these diseases share common etiological bases. Overall, mining of interspecies transcriptome data reveals conserved transcriptomic features of retinas across species and promising applications in both normal retina anatomy and retina pathology.

14.
J Adv Res ; 2023 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-37956862

RESUMO

INTRODUCTION: Global warming is increasing interest in how aquatic animals can adjust their physiological performance and cope with temperature changes. Therefore, understanding the behavioral changes and molecular underpinnings in fish under warming is crucial for both the individual and groups survival. This could provide experimental evidence and resource for evaluating the impact of global warming. OBJECTIVE: Three genetic families of common carp (Cyprinus carpio) were generated. These juveniles were constructed short-term (4 days) and long-term (30 days) warming groups to investigate the effects of warming on behavioral responses and to elucidate the potential underlying mechanisms of warming-driven behavior. METHODS: Behavioral tests were used to explore the effects of short- and long-term exposure to warming on the swimming behavior of C. carpio. Brain transcriptome combined with measurement of nervous system activity was used to further investigated the comprehensive neuromolecular mechanisms under warming. RESULTS: Long-term warming groups had a more significant impact on the decline of swimming behavior in juvenile C. carpio. Furthermore, brain comparative transcriptomic analysis combined with measurement of nervous system activity revealed that genes involved in cytoskeletal organization, mitochondrial regulation, and energy metabolism are major regulators of behavior in the juvenile under warming. Importantly, especially in the long-term warming groups, enrichment analysis of associated gene expression suggested functional alterations of synaptic transmission and signal transduction leading to swimming function impairment in the central nervous system, as revealed by behavioral tests. CONCLUSIONS: Our study provides evidence of the neurogenomic mechanism underlying the decreased swimming activity in juvenile C. carpio under warming. These findings have important implications for understanding the impacts of climate change on aquatic ecosystems and the organisms that inhabit them.

15.
Fish Shellfish Immunol ; 33(3): 459-67, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22659441

RESUMO

Tollip and IRAK-1 are key components of the TLR/IL-1R signaling pathway in mammals, which play crucial roles as mediators of the TLR/IL-1R signal transduction pathways. Although several TLRs have been found in fish, molecular associations, protein-protein interactions or the role of the TLR signaling pathway in infection-induced immunity in fish has received little attention. In this study, Tollip and IRAK-1 sequences of grass carp were isolated from a head kidney cDNA library. Full length transcripts and sequences of promoter regions were obtained by 3' and 5' RACE and genome walking, respectively. Reporter gene-promoter constructs and real-time RT-PCR analysis was used to determine grass carp Tollip and IRAK-1 transcription pattern in tissues. Recombinant proteins were used for antibodies production. Phylogenetically, the grass carp loci clustered with previously reported Tollip and IRAK-1genes, respectively, and their sequences shared the highest identity with the genes of zebrafish (Danio rerio). The promoter region of grass carp Tollip and IRAK-1 proved to be active. After viral infection transcript levels of both loci were upregulated in most immune-related tissues in a time-dependent manner. Using antibodies produced in this study, immunofluorescence analysis indicated that Tollip and IRAK-1 were uniformly distributed and co-localized in the cytoplasm of CIK cells. After viral infection, however, Tollip and IRAK-1 both trended toward the cell membrane. Our results demonstrate the existence of Tollip and IRAK-1 proteins in teleost species, and suggest that Tollip-IRAK-1 complexes are being recruited to receptor complexes after stimulation with virus. These results provide novel insights into the role of the TLR signaling pathway in teleosts, especially the action of teleost Tollip and IRAK-1 and the interaction of these molecules as part of this pathway.


Assuntos
Carpas/genética , Proteínas de Peixes/genética , Rim Cefálico/metabolismo , Quinases Associadas a Receptores de Interleucina-1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transdução de Sinais , Receptores Toll-Like/metabolismo , Sequência de Aminoácidos , Animais , Carpas/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica/veterinária , Quinases Associadas a Receptores de Interleucina-1/química , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reoviridae/fisiologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/veterinária , Infecções por Reoviridae/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA/veterinária , Homologia de Sequência de Aminoácidos
16.
Fish Shellfish Immunol ; 32(6): 1022-31, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22554574

RESUMO

Toll-like receptor 22 (TLR22) is a fish-specific TLR which recognizes double-strand (ds) RNA and participates in the innate immune response through the Toll-IL-1R homology domain-containing adaptor protein 1 (TICAM-1). To further investigate how the innate immune system of teleosts responds to viral infections, we cloned the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) TLR22 (CiTLR22). The complete cDNA sequence of CiTLR22 was 3647 bp and encodes a polypeptide of 954 amino acids. Analysis of the deduced amino acid sequence indicated that CiTLR22 has typical structural features of proteins belonging to the TLR family. These included 17 LRR domains (residues 88-634) and one C-terminal LRR domain (LRR-CT, residues 694-745) in the extracellular region, and a TIR domain (residues 801-944) in the cytoplasmic region. Comparison with homologous proteins showed that the deduced CiTLR22 has the highest sequence identity to common carp TLR22 (82.9%). Genomic DNA of CiTLR22 was obtained by long-distance (Ld) PCR and structure analysis revealed that the CiTLR22 gene is encoded by uninterrupted exons. Reverse transcriptase-PCR (RT-PCR) revealed that CiTLR22 is a non-maternal gene. It is prominently expressed in immune relevant tissues such as spleen and head kidney. Quantitative RT-PCR analysis showed that CiTLR22 transcripts were upregulated significantly in immune relevant tissues and blood following grass carp reovirus (GCRV) infection. In the whole genomic sequence, nine single nucleotide polymorphisms (SNPs) were detected. Seven of them were sited in the coding region, and the other two located in the 5' and 3' untranslated region (UTR) respectively. None of the SNPs was associated with the resistance of grass carp to GCRV. These results suggested a role for CiTLR22 in mediating immune protection against viral infection in grass carp.


Assuntos
Carpas/genética , Carpas/metabolismo , Regulação da Expressão Gênica , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Carpas/embriologia , Clonagem Molecular , Doenças dos Peixes/imunologia , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/veterinária , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Receptores Toll-Like/química , Receptores Toll-Like/imunologia
17.
Front Immunol ; 13: 938326, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36091072

RESUMO

Organisms are colonized by microorganism communities and play a pivotal role in host function by influencing physiology and development. In mammals, bacterial community may alter gonadal maturation and drive sex-specific differences in gene expression and metabolism. However, bacterial microbiota diversity in the gonads of early vertebrates has not been fully elucidated. Here, we focused on the swamp eel (Monopterus albus), which naturally undergoes sex reversal, and systematically analyzed the bacterial microbiota profiles between females and males using 16S rRNA gene sequences. Specifically, the microbial abundance and community diversity of gonads in males were higher than in females. Although Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were characterized as the dominating phyla in ovary and testis, the relative abundance of Firmicutes was significantly higher in males than females. Detailed analysis of the microbial community revealed that Bacilli were the dominant bacteria in ovaries and Clostridium in testes of M. albus. More importantly, we proposed that differences in the microbial composition and distribution between ovaries and testes may be linked to functional categories in M. albus, especially metabolism. These findings represent a unique resource of bacterial community in gonads to facilitate future research about the mechanism of how microbiota influence sex-specific differences and sex reversal in vertebrates.


Assuntos
Microbiota , Smegmamorpha , Animais , Bactérias/metabolismo , Feminino , Masculino , Mamíferos/genética , Ovário , RNA Ribossômico 16S/genética , Smegmamorpha/genética
18.
Life (Basel) ; 12(9)2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36143477

RESUMO

Alternative splicing is an important way of regulating gene functions in eukaryotes. Several key genes involved in sex determination and gonadal differentiation, such as nr5a1 and ddx4, have sex-biased transcripts between males and females, suggesting a potential regulatory role of alternative splicing in gonads. Currently, the sex-specific alternative splicing events and genes have not been comprehensively studied at the genome-wide level in zebrafish. In this study, through global splicing analysis on three independent sets of RNA-seq data from matched zebrafish testes and ovaries, we identified 120 differentially spliced genes shared by the three datasets, most of which haven't been reported before. Functional enrichment analysis showed that the GO terms of mRNA processing, mRNA metabolism and microtubule-based process were strongly enriched. The testis- and ovary-biased alternative splicing genes were identified, and part of them (tp53bp1, tpx2, mapre1a, kif2c, and ncoa5) were further validated by RT-PCR. Sequence characteristics analysis suggested that the lengths, GC contents, and splice site strengths of the alternative exons or introns may have different influences in different types of alternative splicing events. Interestingly, we identified an unexpected high proportion (over 70%) of non-frameshift exon-skipping events, suggesting that in these cases the two protein isoforms derived from alternative splicing may both have functions. Furthermore, as a representative example, we found that the alternative splicing of ncoa5 causes the loss of a conserved RRM domain in the short transcript predominantly produced in testes. Our study discovers novel sex-specific alternative splicing events and genes with high reliabilities in zebrafish testes and ovaries, which would provide attractive targets for follow-up studies to reveal the biological significances of alternative splicing events and genes in sex determination and gonadal differentiation.

19.
Front Immunol ; 13: 872896, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35844551

RESUMO

Various bacterial diseases have caused great economic losses to the high-density and intensive aquaculture industry; however, the pathogenic mechanism underlying the large-scale challenged to caused by many bacteria remain unclear, making the prevention and treatment of these diseases difficult. In the present study, we isolated a bacterial strain from Cyprinus carpio having a typical bacterial disease and named it Cc2021. Through subsequent morphological observations, a regression challenge, biochemical identification, and 16S rRNA gene sequence analysis, we determined Cc2021 to be Plesiomonas shigelloides. Subsequently, we comprehensively investigated the pathogenicity of P. shigelloides in C. carpio through a regression challenge and assessed the underlying the pathogenic mechanism. Mortality results revealed that P. shigelloides is highly pathogenic and infects various tissues throughout the body, resulting in edema of the liver, spleen, and body and head kidneys. Histopathological analysis revealed obvious inflammation, bleeding, and necrosis in the intestine, spleen, and head kidney. The body's immune tissues actively produce complement C3, superoxide dismutase, and lysozyme after a challenge to resist bacterial invasion. With regard to the underlying pathogenesis of P. shigelloides, comparative transcriptome analysis revealed 876 upregulated genes and 828 downregulated genes in the intestine of C. carpio after the challenge. Analysis of differentially expressed unigenes revealed the involvement of major immune pathways, particularly the TNF signaling pathway, interleukin (IL)-17 signaling pathway, and Toll-like receptor signaling pathway. The present study provides new valuable information on the immune system and defense mechanisms of P. shigelloides.


Assuntos
Carpas , Plesiomonas , Animais , Plesiomonas/genética , RNA Ribossômico 16S/genética , Transcriptoma , Virulência
20.
Nat Struct Mol Biol ; 29(1): 21-31, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35013598

RESUMO

Analogous to alternative splicing, alternative polyadenylation (APA) has long been thought to occur independently at proximal and distal polyA sites. Using fractionation-seq, we unexpectedly identified several hundred APA genes in human cells whose distal polyA isoforms are retained in chromatin/nuclear matrix and whose proximal polyA isoforms are released into the cytoplasm. Global metabolic PAS-seq and Nanopore long-read RNA-sequencing provide further evidence that the strong distal polyA sites are processed first and the resulting transcripts are subsequently anchored in chromatin/nuclear matrix to serve as precursors for further processing at proximal polyA sites. Inserting an autocleavable ribozyme between the proximal and distal polyA sites, coupled with a Cleave-seq approach that we describe here, confirms that the distal polyA isoform is indeed the precursor to the proximal polyA isoform. Therefore, unlike alternative splicing, APA sites are recognized independently, and in many cases, in a sequential manner. This provides a versatile strategy to regulate gene expression in mammalian cells.


Assuntos
Poli A/metabolismo , Poliadenilação , Regiões 3' não Traduzidas/genética , Processamento Alternativo/genética , Células HeLa , Humanos , Íntrons/genética , Matriz Nuclear/metabolismo , RNA/metabolismo , RNA Polimerase II/metabolismo
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