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1.
Infect Genet Evol ; 115: 105515, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37866684

RESUMO

Porcine parvovirus (PPV) is an important pathogen causing reproductive disorders in sows, with clinical symptoms including stillbirth, mummified fetuses, embryonic dysplasia and death, and sow infertility. Porcine parvovirus 7 (PPV7) is a recently discovered type of PPV and its widespread distribution and rapid evolution has caused huge economic losses in the pig industry. To investigate the molecular epidemiology of PPV7 in Fujian Province, China, we collected 491 blood samples and 72 tissue samples from diseased pigs in large-scale pig farms across selected areas of Fujian Province from 2019 to 2022. PPV7 infection was determined using real-time quantitative PCR, and positive samples underwent whole-genome amplification, sequencing, and subsequent homology, phylogenetic, and recombination analyses. The PPV7 positive detection rate was 25.73% (145/563) in Fujian Province, among which the positive rate of blood and tissue samples was 26.47% (130/491) and 20.83% (15/72), respectively. The nucleotide sequence homology among the 29 PPV7 whole-genome sequences obtained in this study was 90.0%-97.2%, whereas that with 128 reference strains from China and other countries was 88.9%-98.1%. Six strains had partial nucleotide deletions or insertions. Phylogenetic analysis based on the whole-genome sequences classified the 29 PPV7 strains and 128 reference strains into eight subtypes (PPV7a-PPV7h), and PPV7h was the predominant subtype in Fujian Province. Recombination analysis revealed evidence of inferred recombination events in the genomes of four strains. This study provides significant insights into the molecular characteristics of PPV7 in Fujian Province and serves as a crucial foundation for further advancements in PPV7 prevention and control strategies.


Assuntos
Infecções por Parvoviridae , Parvovirus Suíno , Doenças dos Suínos , Suínos , Animais , Feminino , Doenças dos Suínos/epidemiologia , Parvovirus Suíno/genética , Filogenia , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/veterinária , Homologia de Sequência do Ácido Nucleico , China/epidemiologia
2.
Virus Genes ; 59(1): 109-120, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36383275

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) has continuously mutated since its first isolation in China in 1996, leading to difficulties in infection prevention and control. Infections caused by PRRSV-2 strains are the main epidemic strains in China, as determined by phylogenetic analysis. In this study, we focused on the prevalence and genetic variations of the non-structural protein 4 (NSP4) from PRRSV-2 over the past 20 years in China. The fundamental biological properties of the NSP4 were predicted, and an analysis and comparison of NSP4 homology at the nucleotide and amino acid levels was conducted using 123 PRRSV-2 strains. The predicted molecular weight of the NSP4 protein was determined to be 21.1 kDa, and it was predicted to be a stable hydrophobic protein that lacks a signal peptide. NSP4 from different strains exhibited a high degree of amino acid (85.8-100%) and nucleotide sequence homology (81.0-100%). Multiple amino acid substitutions were identified in NSP4 among 15 representative PRRSV-2 strains. Phylogenetic analysis showed that the lineage 8 and 1 strains, the most prevalent strains in China, were indifferent clades with a long genetic distance. This analysis will help fully elucidate the parameters of the PRRSV NSP4 epidemic in China to lay a foundation for adequate understanding of the function of NSP4. Genetic information results from the accumulation of conserved and non-conserved sequences. The high conservation of the NSP4 gene determines the most basic life traits and functions of PRRSV. Analyzing the spatial structure of NSP4 protein and studying the genetic evolution of NSP4 not only provide the theoretical basis for how NSP4 participates in the regulation of the innate response of the host but also provide a target for genetic manipulation and a reasonable target molecule and structure for new drug molecules.


Assuntos
Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Suínos , Animais , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , Síndrome Respiratória e Reprodutiva Suína/genética , Filogenia , Homologia de Sequência do Ácido Nucleico , Aminoácidos , China/epidemiologia , Variação Genética
3.
Nucleic Acids Res ; 50(5): 2509-2521, 2022 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-35234938

RESUMO

Upon SARS-CoV-2 infection, viral intermediates specifically activate the IFN response through MDA5-mediated sensing and accordingly induce ADAR1 p150 expression, which might lead to viral A-to-I RNA editing. Here, we developed an RNA virus-specific editing identification pipeline, surveyed 7622 RNA-seq data from diverse types of samples infected with SARS-CoV-2, and constructed an atlas of A-to-I RNA editing sites in SARS-CoV-2. We found that A-to-I editing was dynamically regulated, varied between tissue and cell types, and was correlated with the intensity of innate immune response. On average, 91 editing events were deposited at viral dsRNA intermediates per sample. Moreover, editing hotspots were observed, including recoding sites in the spike gene that affect viral infectivity and antigenicity. Finally, we provided evidence that RNA editing accelerated SARS-CoV-2 evolution in humans during the epidemic. Our study highlights the ability of SARS-CoV-2 to hijack components of the host antiviral machinery to edit its genome and fuel its evolution, and also provides a framework and resource for studying viral RNA editing.


Assuntos
COVID-19/imunologia , Imunidade Inata/imunologia , Edição de RNA/imunologia , SARS-CoV-2/imunologia , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Adenosina Desaminase/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Sequência de Bases , Sítios de Ligação/genética , COVID-19/genética , COVID-19/virologia , Evolução Molecular , Expressão Gênica/imunologia , Humanos , Imunidade Inata/genética , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Mutação , Ligação Proteica , Edição de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Homologia de Sequência do Ácido Nucleico , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
Int J Mol Sci ; 23(3)2022 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-35163686

RESUMO

Three Dysteria species, D. crassipes Claparède & Lachmann, 1859; D. brasiliensis Faria et al., 1922; and D. paracrassipes n. sp., were collected from subtropical coastal waters of the East China Sea, near Ningbo, China. The three species were studied based on their living morphology, infraciliature, and molecular data. The new species D. paracrassipes n. sp. is very similar to D. crassipes in most morphological features except the preoral kinety, which is double-rowed in the new species (vs. single-rowed in D. crassipes). The difference in the small ribosomal subunit sequences (SSU rDNA) between these two species is 56 bases, supporting the establishment of the new species. The Ningbo population of D. crassipes is highly similar in morphology to other known populations. Nevertheless, the SSU rDNA sequences of these populations are very different, indicating high genetic diversity and potentially cryptic species. Dysteria brasiliensis is cosmopolitan with many described populations worldwide and four deposited SSU rDNA sequences. The present work supplies morphological and molecular information from five subtropical populations of D. brasiliensis that bear identical molecular sequences but show significant morphological differences. The findings of this study provide an opportunity to improve understanding of the morphological and genetic diversity of ciliates.


Assuntos
Cilióforos/classificação , Cilióforos/genética , Filogenia , Sequência de Bases , China , DNA Ribossômico/genética , Geografia , Funções Verossimilhança , RNA Ribossômico/genética , Subunidades Ribossômicas Menores/genética , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie
5.
Mol Immunol ; 143: 58-67, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35042118

RESUMO

Interleukin 12 (IL-12) binds its receptor complex of IL-12 receptor beta 1 (IL-12Rß1) and IL-12Rß2 to transduce cellular signaling in mammals. In teleosts, the function of Il-12 is drawing increasing attention, but molecular and functional features of Il-12 receptors remain obscure. Especially, the existence of multiple Il-12 isoforms in some fish species elicits the requirement to clarify their receptors. In this study, we isolated three cDNA sequences as Il-12 receptor candidates from grass carp, entitled as grass carp Il-12rß1 (gcIl-12rß1), gcIl-12rß2a and gcIl-12rß2b. In silico analysis showed that gcIl-12rß1 and gcIl-12rß2a shared the conserved gene locus and similar structure characteristics with their orthologues of zebrafish, frog, chicken, mouse and human, respectively. However, the Il-12rß2b of grass carp and zebrafish was similar to IL-27Ra in non-fish species. Further locally installed BLAST and gene synteny analysis uncovered three gcIl-12 receptors being single copied genes. Tissue distribution assay revealed that gcil12rß1 and gcil12rß2a transcripts were predominantly expressed in head kidney, differing from the even distribution of gcil12rß2b transcripts in all detected tissues. Subsequently, the binding ability and antagonistic effects of recombinant extracellular region of gcIl-12rß1 with recombinant grass carp Il-12 (rgcIl-12) isoforms were explored, providing functional evidence of the newly cloned gcIl-12rß1 being genuine orthologues of mammalian IL-12Rß1. Moreover, our data showed that gcIl-12rß1 and gcIl-12rß2a but not gcIl-12rß1 and gcIl-12rß2b mediated the effects of rgcIl-12 isoforms on ifn-γ promoter activity, thereby revealing Il-12 receptor signaling in fish. These results identified grass carp Il-12 receptors, thereby advancing our understanding of Il-12 isoform signaling in fish.


Assuntos
Carpas/metabolismo , Subunidade beta 1 de Receptor de Interleucina-12/metabolismo , Subunidade beta 2 de Receptor de Interleucina-12/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Perfilação da Expressão Gênica , Humanos , Subunidade beta 1 de Receptor de Interleucina-12/química , Subunidade beta 1 de Receptor de Interleucina-12/genética , Subunidade beta 2 de Receptor de Interleucina-12/química , Subunidade beta 2 de Receptor de Interleucina-12/genética , Filogenia , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência do Ácido Nucleico , Sintenia/genética
6.
Mol Med Rep ; 25(3)2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35039872

RESUMO

Long non­coding RNAs (lncRNAs) feature prominently in pancreatic carcinoma progression. The present study aimed to clarify the biological functions, clinical significance and underlying mechanism of lncRNA CTBP1 antisense RNA 2 (CTBP1­AS2) in pancreatic carcinoma. Reverse transcription­quantitative PCR was performed to assess the expression levels of CTBP1­AS2, microRNA (miR)­141­3p and ubiquitin­specific protease 22 (USP22) mRNA in pancreatic carcinoma tissues and cell lines. Western blotting was used to examine USP22 protein expression in pancreatic carcinoma cell lines. Loss­of­function experiments were used to analyze the regulatory effects of CTBP1­AS2 on proliferation, apoptosis, migration and invasion of pancreatic carcinoma cells. Dual­luciferase reporter assay was used to examine the binding relationship between CTBP1­AS2 and miR­141­3p, as well as between miR­141­3p and USP22. It was demonstrated that CTBP1­AS2 expression was markedly increased in pancreatic carcinoma tissues and cell lines. High CTBP1­AS2 expression was associated with advanced clinical stage and lymph node metastasis of patients. Functional experiments confirmed that knocking down CTBP1­AS2 significantly inhibited pancreatic carcinoma cell proliferation, migration and invasion, and promoted cell apoptosis. In terms of mechanism, it was found that CTBP1­AS2 adsorbed miR­141­3p as a molecular sponge to upregulate the expression level of USP22. In conclusion, lncRNA CTBP1­AS2 may be involved in pancreatic carcinoma progression by regulating miR­141­3p and USP22 expressions; in addition, CTBP1­AS2 may be a diagnostic biomarker and treatment target for pancreatic carcinoma.


Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Pancreáticas/genética , RNA Longo não Codificante/genética , Ubiquitina Tiolesterase/genética , Apoptose/genética , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Interferência de RNA , Homologia de Sequência do Ácido Nucleico , Ubiquitina Tiolesterase/metabolismo , Regulação para Cima
7.
Biochim Biophys Acta Mol Basis Dis ; 1868(2): 166291, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34662705

RESUMO

OBJECTIVES: To investigate in silico the presence of nucleotide sequence complementarity between the RNA genome of Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) and human non-coding (nc)RNA genes. METHODS: The FASTA sequence (NC_045512.2) of each of the 11 SARS-CoV-2 isolate Wuhan-Hu-1 genes was retrieved from NCBI.nlm.nih.gov/gene and the Ensembl.org library interrogated for any base-pair match with human ncRNA genes. SARS-CoV-2 gene-matched human ncRNAs were screened for functional activity using bioinformatic analysis. Finally, associations between identified ncRNAs and human diseases were searched in GWAS databases. RESULTS: A total of 252 matches were found between the nucleotide sequence of SARS-CoV-2 genes and human ncRNAs. With the exception of two small nuclear RNAs, all of them were long non-coding (lnc)RNAs expressed mainly in testis and central nervous system under physiological conditions. The percentage of alignment ranged from 91.30% to 100% with a mean nucleotide alignment length of 17.5 ± 2.4. Thirty-three (13.09%) of them contained predicted R-loop forming sequences, but none of these intersected the complementary sequences of SARS-CoV-2. However, in 31 cases matches fell on ncRNA regulatory sites, whose adjacent coding genes are mostly involved in cancer, immunological and neurological pathways. Similarly, several polymorphic variants of detected non-coding genes have been associated with neuropsychiatric and proliferative disorders. CONCLUSION: This pivotal in silico study shows that SARS-CoV-2 genes have Watson-Crick nucleotide complementarity to human ncRNA sequences, potentially disrupting ncRNA epigenetic control of target genes. It remains to be elucidated whether this could result in the development of human disease in the long term.


Assuntos
COVID-19/genética , COVID-19/virologia , RNA não Traduzido/genética , SARS-CoV-2/genética , Sequência de Bases , Epigênese Genética , Genes Virais , Humanos , Neoplasias/genética , RNA Longo não Codificante/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
8.
Lab Invest ; 102(1): 38-47, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34326457

RESUMO

Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide. Dysregulation of circular RNAs (circRNAs) appears to be a critical factor in CRC progression. However, mechanistic studies delineating the role of circRNAs in CRC remain limited. In this study, qRT-PCR and western blot assays were used to measure the expression of genes and proteins. Migration, invasion, proliferation, and apoptosis were examined by wound-healing, transwell, CCK-8, colony formation, and flow cytometry assays, respectively. Molecular interactions were validated by a dual-luciferase report system. A xenograft animal model was established to examine in vivo tumor growth and lung metastasis. Our data indicated that circN4BP2L2 expression was increased in CRC tissues and cell lines. Notably, inhibition of circN4BP2L2 effectively inhibited proliferation, migration, and invasion of LoVo cells, and inhibited tumor growth and metastasis in vivo, whereas the forced expression of circN4BP2L2 facilitated the proliferation, migration, and invasion of HT-29 cells. Mechanistic studies revealed that circN4BP2L2 acted as a molecular sponge of miR-340-5p to competitively promote CXCR4 expression. Furthermore, inhibition of miR-340-5p reversed the anti-cancer effects of circN4BP2L2 or CXCR4 silencing. Our data indicated an oncogenic role of circN4BP2L2 in CRC via regulation of the miR-340-5p/CXCR4 axis, which may be a promising biomarker and target for CRC treatment.


Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , RNA Circular/genética , Receptores CXCR4/genética , Animais , Apoptose/genética , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Células HCT116 , Células HT29 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Terapêutica com RNAi/métodos , Homologia de Sequência do Ácido Nucleico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
9.
Genes Genomics ; 44(1): 39-51, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34455578

RESUMO

BACKGROUND: The net ammonium fluxes differ among the different root zones of Populus, but the physiological and microRNA regulatory mechanisms are unclear. OBJECTIVE: To elucidate the physiological and miRNA regulatory mechanisms, we investigated the two root zones displaying significant differences in net NH4+ effluxes of P. × canescens. METHODS: Populus plantlets were cultivated with 500 µM NH4Cl for 10 days. Six plants were randomly selected to determine the net NH4+ fluxes using a noninvasive microtest technique. High-throughput sequencing were used to determine the dynamic expression profile of miRNA among the different root zones of Populus. RESULTS: Net NH4+ efflux in zone I (from 0 to 40 mm from the root apex) was - 19.64 pmol cm-2 s-1 and in zone II (from 40 to 80 mm) it was - 43.96 pmol cm-2 s-1. The expression of eleven miRNAs was significantly upregulated, whereas fifteen miRNAs were downregulated. Moreover, eighty-eight target genes of the significantly differentially expressed miRNAs were identified in root zone II compared with zone I. Particularly, ptc-miR171a/b/e and their target, SCL6, were found to be important for the difference in net NH4+ effluxes in the two root zones. Moreover, the expression of the target of ptc-miR169d, NFYA3 was upregulated in root zone II compared with root zone I, contributing to increased NH4+ efflux and decreased NH4+ assimilation in root zone II. CONCLUSION: These results indicate that miRNAs regulate the expression levels of their target genes and thus play key roles in net NH4+ fluxes and NH4+ assimilation in different poplar root zones.


Assuntos
Compostos de Amônio/metabolismo , Regulação da Expressão Gênica de Plantas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Raízes de Plantas/genética , Populus/genética , Sequência de Bases , Nitrogênio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Populus/metabolismo , RNA de Plantas/genética , RNA-Seq/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Transcriptoma/genética
10.
Nucleic Acids Res ; 50(D1): D333-D339, 2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-34551440

RESUMO

Resolving the spatial distribution of the transcriptome at a subcellular level can increase our understanding of biology and diseases. To facilitate studies of biological functions and molecular mechanisms in the transcriptome, we updated RNALocate, a resource for RNA subcellular localization analysis that is freely accessible at http://www.rnalocate.org/ or http://www.rna-society.org/rnalocate/. Compared to RNALocate v1.0, the new features in version 2.0 include (i) expansion of the data sources and the coverage of species; (ii) incorporation and integration of RNA-seq datasets containing information about subcellular localization; (iii) addition and reorganization of RNA information (RNA subcellular localization conditions and descriptive figures for method, RNA homology information, RNA interaction and ncRNA disease information) and (iv) three additional prediction tools: DM3Loc, iLoc-lncRNA and iLoc-mRNA. Overall, RNALocate v2.0 provides a comprehensive RNA subcellular localization resource for researchers to deconvolute the highly complex architecture of the cell.


Assuntos
Bases de Dados de Ácidos Nucleicos , RNA não Traduzido/genética , Software , Transcriptoma , Animais , Sequência de Bases , Compartimento Celular , Conjuntos de Dados como Assunto , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Internet , Camundongos , Anotação de Sequência Molecular , RNA não Traduzido/classificação , RNA não Traduzido/metabolismo , Ratos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
11.
Nucleic Acids Res ; 49(22): 12820-12835, 2021 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-34871419

RESUMO

In Escherichia coli, the replication initiator DnaA oscillates between an ATP- and an ADP-bound state in a cell cycle-dependent manner, supporting regulation for chromosome replication. ATP-DnaA cooperatively assembles on the replication origin using clusters of low-affinity DnaA-binding sites. After initiation, DnaA-bound ATP is hydrolyzed, producing initiation-inactive ADP-DnaA. For the next round of initiation, ADP-DnaA binds to the chromosomal locus DARS2, which promotes the release of ADP, yielding the apo-DnaA to regain the initiation activity through ATP binding. This DnaA reactivation by DARS2 depends on site-specific binding of IHF (integration host factor) and Fis proteins and IHF binding to DARS2 occurs specifically during pre-initiation. Here, we reveal that Fis binds to an essential region in DARS2 specifically during pre-initiation. Further analyses demonstrate that ATP-DnaA, but not ADP-DnaA, oligomerizes on a cluster of low-affinity DnaA-binding sites overlapping the Fis-binding region, which competitively inhibits Fis binding and hence the DARS2 activity. DiaA (DnaA initiator-associating protein) stimulating ATP-DnaA assembly enhances the dissociation of Fis. These observations lead to a negative feedback model where the activity of DARS2 is repressed around the time of initiation by the elevated ATP-DnaA level and is stimulated following initiation when the ATP-DnaA level is reduced.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação/genética , Ciclo Celular/genética , Cromossomos Bacterianos/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/genética , Retroalimentação Fisiológica , Fatores Hospedeiros de Integração/genética , Fatores Hospedeiros de Integração/metabolismo , Modelos Genéticos , Ligação Proteica , Origem de Replicação/genética , Homologia de Sequência do Ácido Nucleico
12.
PLoS One ; 16(12): e0261215, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34914734

RESUMO

Dehydration Responsive Element Binding (DREB) regulates the expression of numerous stress-responsive genes, and hence plays a pivotal role in abiotic stress responses and tolerance in plants. The study aimed to develop a complete overview of the cis-acting regulatory elements (CAREs) present in S. tuberosum DREB gene promoters. A total of one hundred and four (104) cis-regulatory elements (CREs) were identified from 2.5kbp upstream of the start codon (ATG). The in-silico promoter analysis revealed variable sets of cis-elements and functional diversity with the predominance of light-responsive (30%), development-related (20%), abiotic stress-responsive (14%), and hormone-responsive (12%) elements in StDREBs. Among them, two light-responsive elements (Box-4 and G-box) were predicted in 64 and 61 StDREB genes, respectively. Two development-related motifs (AAGAA-motif and as-1) were abundant in StDREB gene promoters. Most of the DREB genes contained one or more Myeloblastosis (MYB) and Myelocytometosis (MYC) elements associated with abiotic stress responses. Hormone-responsive element i.e. ABRE was found in 59 out of 66 StDREB genes, which implied their role in dehydration and salinity stress. Moreover, six proteins were chosen corresponding to A1-A6 StDREB subgroups for secondary structure analysis and three-dimensional protein modeling followed by model validation through PROCHECK server by Ramachandran Plot. The predicted models demonstrated >90% of the residues in the favorable region, which further ensured their reliability. The present study also anticipated pocket binding sites and disordered regions (DRs) to gain insights into the structural flexibility and functional annotation of StDREB proteins. The protein association network determined the interaction of six selected StDREB proteins with potato proteins encoded by other gene families such as MYB and NAC, suggesting their similar functional roles in biological and molecular pathways. Overall, our results provide fundamental information for future functional analysis to understand the precise molecular mechanisms of the DREB gene family in S. tuberosum.


Assuntos
Regiões Promotoras Genéticas/genética , Solanum tuberosum/genética , Fatores de Transcrição/genética , Arabidopsis/genética , Desidratação/genética , Secas , Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Estudo de Associação Genômica Ampla/métodos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/genética , Estresse Salino/genética , Homologia de Sequência do Ácido Nucleico , Solanum tuberosum/crescimento & desenvolvimento , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo
13.
Front Immunol ; 12: 766719, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34917085

RESUMO

The human SFTPA1 and SFTPA2 genes encode the surfactant protein A1 (SP-A1) and SP-A2, respectively, and they have been identified with significant genetic and epigenetic variability including sequence, deletion/insertions, and splice variants. The surfactant proteins, SP-A1 and SP-A2, and their corresponding variants play important roles in several processes of innate immunity as well in surfactant-related functions as reviewed elsewhere [1]. The levels of SP-A have been shown to differ among individuals both under baseline conditions and in response to various agents or disease states. Moreover, a number of agents have been shown to differentially regulate SFTPA1 and SFTPA2 transcripts. The focus in this review is on the differential regulation of SFTPA1 and SFTPA2 with primary focus on the role of 5' and 3' untranslated regions (UTRs) and flanking sequences on this differential regulation as well molecules that may mediate the differential regulation.


Assuntos
Variação Genética/imunologia , Imunidade Inata/imunologia , Proteína A Associada a Surfactante Pulmonar/imunologia , Transcriptoma/imunologia , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/imunologia , Regiões 5' não Traduzidas/genética , Regiões 5' não Traduzidas/imunologia , Sequência de Bases , Variação Genética/genética , Humanos , Imunidade Inata/genética , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/metabolismo , Homologia de Sequência do Ácido Nucleico , Transcriptoma/genética
14.
Front Immunol ; 12: 770055, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34868028

RESUMO

Circular RNAs (circRNAs) act as essential regulators in many biological processes, especially in mammalian immune response. Nonetheless, the functions and mechanisms of circRNAs in the invertebrate immune system are largely unclarified. In our previous work, 261 differentially expressed circRNAs potentially related to the development of Apostichopus japonicus skin ulceration syndrome (SUS), which is a major problem restricting the sea cucumber breeding industry, were identified by genome-wide screening. In this study, via miRanda analysis, both circRNA75 and circrRNA72 were shown to share the miR-200 binding site, a key microRNA in the SUS. The two circRNAs were verified to be increased significantly in LPS-exposed primary coelomocytes, similar to the results of circRNA-seq in sea cucumber under Vibrio splendidus-challenged conditions. A dual-luciferase assay indicated that both circRNA75 and circRNA72 could bind miR-200 in vivo, in which circRNA75 had four binding sites of miR-200 and only one for circRNA72. Furthermore, we found that miR-200 could bind the 3'-UTR of Toll interacting protein (Tollip) to negatively mediate the expression of Tollip. Silencing Tollip increased primary coelomocyte apoptosis. Consistently, inference of circRNA75 and circRNA72 could also downregulate Tollip expression, thereby increasing the apoptosis of primary coelomocytes, which could be blocked by miR-200 inhibitor treatment. Moreover, the rate of si-circRNA75-downregulated Tollip expression was higher than that of si-circRNA72 under an equivalent amount. CircRNA75 and circRNA72 suppressed coelomocyte apoptosis by sponging miR-200 to promote Tollip expression. The ability of circRNA to adsorb miRNA might be positively related to the number of binding sites for miRNA.


Assuntos
Apoptose/genética , Sistema Digestório/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , MicroRNAs/genética , RNA Circular/genética , Stichopus/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Células Cultivadas , Sistema Digestório/citologia , Sistema Digestório/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Fagócitos/efeitos dos fármacos , Fagócitos/imunologia , Fagócitos/metabolismo , Homologia de Sequência do Ácido Nucleico , Stichopus/imunologia , Stichopus/virologia , Vibrio/imunologia , Vibrio/fisiologia
15.
Nucleic Acids Res ; 49(22): 12912-12928, 2021 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-34850097

RESUMO

Micro RNAs (miRNAs) are processed from precursor RNA molecules with precisely defined secondary stem-loop structures. ARGONAUTE1 (AGO1) is the main executor component of miRNA pathway and its expression is controlled via the auto-regulatory feedback loop activity of miR168 in plants. Previously we have shown that AGO1 loading of miR168 is strongly restricted leading to abundant cytoplasmic accumulation of AGO-unbound miR168. Here, we report, that intrinsic RNA secondary structure of MIR168a precursor not only defines the processing of miR168, but also precisely adjusts AGO1 loading efficiency determining the biologically active subset of miR168 pool. Our results show, that modification of miRNA duplex structure of MIR168a precursor fragment or expression from artificial precursors can alter the finely adjusted loading efficiency of miR168. In dcl1-9 mutant where, except for miR168, production of most miRNAs is severely reduced this mechanism ensures the elimination of unloaded AGO1 proteins via enhanced AGO1 loading of miR168. Based on this data, we propose a new competitive loading mechanism model for miR168 action: the miR168 surplus functions as a molecular buffer for controlled AGO1 loading continuously adjusting the amount of AGO1 protein in accordance with the changing size of the cellular miRNA pool.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Argonautas/genética , Homeostase/genética , MicroRNAs/genética , Interferência de RNA , Complexo de Inativação Induzido por RNA/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/metabolismo , Sequência de Bases , Regulação da Expressão Gênica de Plantas , MicroRNAs/metabolismo , Mutação , Plantas Geneticamente Modificadas , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico
16.
Nucleic Acids Res ; 49(21): 12332-12347, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34755863

RESUMO

In all cells, DNA topoisomerases dynamically regulate DNA supercoiling allowing essential DNA processes such as transcription and replication to occur. How this complex system emerged in the course of evolution is poorly understood. Intriguingly, a single horizontal gene transfer event led to the successful establishment of bacterial gyrase in Archaea, but its emergent function remains a mystery. To better understand the challenges associated with the establishment of pervasive negative supercoiling activity, we expressed the gyrase of the bacterium Thermotoga maritima in a naïve archaeon Thermococcus kodakarensis which naturally has positively supercoiled DNA. We found that the gyrase was catalytically active in T. kodakarensis leading to strong negative supercoiling of plasmid DNA which was stably maintained over at least eighty generations. An increased sensitivity of gyrase-expressing T. kodakarensis to ciprofloxacin suggested that gyrase also modulated chromosomal topology. Accordingly, global transcriptome analyses revealed large scale gene expression deregulation and identified a subset of genes responding to the negative supercoiling activity of gyrase. Surprisingly, the artificially introduced dominant negative supercoiling activity did not have a measurable effect on T. kodakarensis growth rate. Our data suggest that gyrase can become established in Thermococcales archaea without critically interfering with DNA transaction processes.


Assuntos
Proteínas de Bactérias/genética , DNA Girase/genética , DNA Arqueal/genética , DNA Super-Helicoidal/genética , Temperatura Alta , Thermococcus/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biocatálise , Ciprofloxacina/farmacologia , DNA Girase/metabolismo , DNA Arqueal/metabolismo , DNA Super-Helicoidal/metabolismo , Regulação da Expressão Gênica em Archaea/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Microscopia Confocal , Plasmídeos/genética , Plasmídeos/metabolismo , Homologia de Sequência do Ácido Nucleico , Thermococcus/efeitos dos fármacos , Thermococcus/metabolismo , Thermotoga maritima/enzimologia , Thermotoga maritima/genética
17.
Sci Rep ; 11(1): 19161, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34580386

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with fatal pulmonary fibrosis. Small interfering RNAs (siRNAs) can be developed to induce RNA interference against SARS-CoV-2, and their susceptible target sites can be inferred by Argonaute crosslinking immunoprecipitation sequencing (AGO CLIP). Here, by reanalysing AGO CLIP data in RNA viruses, we delineated putative AGO binding in the conserved non-structural protein 12 (nsp12) region encoding RNA-dependent RNA polymerase (RdRP) in SARS-CoV-2. We utilised the inferred AGO binding to optimise the local RNA folding parameter to calculate target accessibility and predict all potent siRNA target sites in the SARS-CoV-2 genome, avoiding sequence variants. siRNAs loaded onto AGO also repressed seed (positions 2-8)-matched transcripts by acting as microRNAs (miRNAs). To utilise this, we further screened 13 potential siRNAs whose seed sequences were matched to known antifibrotic miRNAs and confirmed their miRNA-like activity. A miR-27-mimicking siRNA designed to target the nsp12 region (27/RdRP) was validated to silence a synthesised nsp12 RNA mimic in lung cell lines and function as an antifibrotic miR-27 in regulating target transcriptomes related to TGF-ß signalling. siRNA sequences with an antifibrotic miRNA-like activity that could synergistically treat COVID-19 are available online ( http://clip.korea.ac.kr/covid19 ).


Assuntos
Proteínas Argonautas/genética , COVID-19/prevenção & controle , MicroRNAs/genética , RNA Interferente Pequeno/genética , SARS-CoV-2/genética , Células A549 , Proteínas Argonautas/metabolismo , Sequência de Bases , Sítios de Ligação/genética , COVID-19/virologia , Linhagem Celular , RNA-Polimerase RNA-Dependente de Coronavírus/genética , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Perfilação da Expressão Gênica/métodos , Células HeLa , Humanos , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Interferência de RNA , RNA-Seq/métodos , SARS-CoV-2/fisiologia , Homologia de Sequência do Ácido Nucleico
18.
Nature ; 597(7876): 426-429, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34471288

RESUMO

Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3' single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms.


Assuntos
DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Biológicos , Recombinases Rec A/metabolismo , Reparo de DNA por Recombinação , Homologia de Sequência do Ácido Nucleico , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/metabolismo , Fatores de Tempo
19.
J Comput Biol ; 28(10): 961-974, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34491118

RESUMO

We extend the popular Jukes-Cantor evolution model and calculate the probability of an orthologous nucleotide sequence set [a reference sequence (B1) stays with the other sequences (B-1)], where the sequence evolution [from a last common ancestral sequence (ɑ)] follows the (prospective) Poisson process with the overall event rate λ prorated among mutation types (nucleotide/codon substitution, insertion, and deletion) and sites along each sequence. The corresponding retrospective process (reversing the prospective process) facilitates developing algorithms to calculate the marginal probability [Pr(B1)] (Monte Carlo integration) and sample ɑ (given B1). We calculate probability Pr(B-1|ɑ) based on the identified events (during "ɑ→B-1") from pairwise sequence alignment to implement Pr(B-1|B1) calculation (Monte Carlo integration). Event queue sampling and probability magnifiers are used to improve the computational efficiency when the number of events is large. We finally test our procedure on both simulated and recently studied hexapod transcriptome data (Brandt et al.), where each asexual lineage pairs with its closest related sexual lineage. Rate estimates (for Phasmatodea and Zygentoma) and model comparison indicate that the asexual lineages likely mutate several times faster than their sexual relatives.


Assuntos
Biologia Computacional/métodos , Insetos/classificação , Algoritmos , Animais , Evolução Molecular , Insetos/genética , Modelos Genéticos , Método de Monte Carlo , Neópteros/genética , Filogenia , Distribuição de Poisson , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico
20.
Nat Commun ; 12(1): 5557, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34548488

RESUMO

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs.


Assuntos
Nadadeiras de Animais/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Botões de Extremidades/metabolismo , Nadadeiras de Animais/citologia , Nadadeiras de Animais/crescimento & desenvolvimento , Animais , Sequência de Bases , Evolução Biológica , Boidae , Bovinos , Galinhas , Embrião de Mamíferos , Embrião não Mamífero , Iguanas , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Botões de Extremidades/citologia , Botões de Extremidades/crescimento & desenvolvimento , Camundongos , Camundongos Transgênicos , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Coelhos , Genética Reversa/métodos , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Tubarões , Transdução de Sinais , Suínos
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