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1.
Viruses ; 13(7)2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34372533

RESUMO

Approximately 240 million people are chronically infected with hepatitis B virus (HBV), despite four decades of effective HBV vaccination. During chronic infection, HBV forms two distinct templates responsible for viral transcription: (1) episomal covalently closed circular (ccc)DNA and (2) host genome-integrated viral templates. Multiple ubiquitous and liver-specific transcription factors are recruited onto these templates and modulate viral gene transcription. This review details the latest developments in antivirals that inhibit HBV gene transcription or destabilize viral transcripts. Notably, nuclear receptor agonists exhibit potent inhibition of viral gene transcription from cccDNA. Small molecule inhibitors repress HBV X protein-mediated transcription from cccDNA, while small interfering RNAs and single-stranded oligonucleotides result in transcript degradation from both cccDNA and integrated templates. These antivirals mediate their effects by reducing viral transcripts abundance, some leading to a loss of surface antigen expression, and they can potentially be added to the arsenal of drugs with demonstrable anti-HBV activity. Thus, these candidates deserve special attention for future repurposing or further development as anti-HBV therapeutics.


Assuntos
Vírus da Hepatite B/genética , Hepatite B/prevenção & controle , Transcrição Genética/genética , Antivirais/farmacologia , DNA Circular/metabolismo , DNA Viral/genética , Hepatite B/tratamento farmacológico , Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Humanos , Fígado/virologia , RNA Interferente Pequeno/metabolismo , Transcrição Genética/fisiologia , Integração Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
2.
Nat Commun ; 12(1): 4941, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400639

RESUMO

Plant small RNAs are important regulatory elements that fine-tune gene expression and maintain genome integrity by silencing transposons. Reproductive organs of monocots produce abundant phased, small interfering RNAs (phasiRNAs). The 21-nt reproductive phasiRNAs triggered by miR2118 are highly enriched in pre-meiotic anthers, and have been found in multiple eudicot species, in contrast with prior reports of monocot specificity. The 24-nt reproductive phasiRNAs are triggered by miR2275, and are highly enriched during meiosis in many angiosperms. Here, we report the widespread presence of the 21-nt reproductive phasiRNA pathway in eudicots including canonical and non-canonical microRNA (miRNA) triggers of this pathway. In eudicots, these 21-nt phasiRNAs are enriched in pre-meiotic stages, a spatiotemporal distribution consistent with that of monocots and suggesting a role in anther development. Although this pathway is apparently absent in well-studied eudicot families including the Brassicaceae, Solanaceae and Fabaceae, our work in eudicots supports an earlier singular finding in spruce, a gymnosperm, indicating that the pathway of 21-nt reproductive phasiRNAs emerged in seed plants and was lost in some lineages.


Assuntos
Magnoliopsida/metabolismo , Nucleotídeos/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/metabolismo , Sementes/metabolismo , Fragaria/genética , Fragaria/metabolismo , Regulação da Expressão Gênica de Plantas , Meiose , MicroRNAs/genética , Filogenia , Picea/genética , Proteínas de Plantas/genética , RNA de Cadeia Dupla/metabolismo , Solanaceae/metabolismo , Transcriptoma
3.
Am J Hum Genet ; 108(9): 1710-1724, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34450031

RESUMO

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.


Assuntos
Osso e Ossos/metabolismo , Complexo I de Proteína do Envoltório/genética , Proteína Coatomer/genética , Deficiências do Desenvolvimento/genética , Deficiência Intelectual/genética , Osteoporose/genética , Animais , Ácido Ascórbico/farmacologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Criança , Pré-Escolar , Complexo I de Proteína do Envoltório/deficiência , Proteína Coatomer/química , Proteína Coatomer/deficiência , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/patologia , Embrião não Mamífero , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Complexo de Golgi , Haploinsuficiência , Humanos , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Masculino , Camundongos , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Osteoporose/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Índice de Gravidade de Doença , Peixe-Zebra
4.
N Engl J Med ; 385(8): 707-719, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34347949

RESUMO

BACKGROUND: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility. METHODS: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing. RESULTS: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in PNLDC1: the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying PNLDC1 mutations. CONCLUSIONS: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.).


Assuntos
Azoospermia/genética , Exorribonucleases/genética , Infertilidade Masculina/genética , Meiose/fisiologia , Mutação , RNA Interferente Pequeno/metabolismo , Testículo/patologia , Adulto , Azoospermia/fisiopatologia , Biópsia , Expressão Gênica , Humanos , Masculino , Fenótipo , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/ultraestrutura , Análise de Sequência de RNA , Testículo/metabolismo , Sequenciamento Completo do Exoma
5.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361779

RESUMO

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.


Assuntos
Cavéolas/efeitos dos fármacos , Colesterol/química , Células Endoteliais/efeitos dos fármacos , Lipossomos/química , Microdomínios da Membrana/efeitos dos fármacos , Transfecção/métodos , Animais , Cavéolas/química , Cavéolas/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Transformada , Colesterol/metabolismo , Clatrina/metabolismo , DNA/química , DNA/metabolismo , Endocitose/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Filipina/química , Filipina/farmacologia , Expressão Gênica , Lipossomos/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Nistatina/química , Nistatina/farmacologia , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacologia , Pinocitose/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos
6.
Science ; 373(6551): 231-236, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244417

RESUMO

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.


Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Interferência de RNA , Vírus de RNA/fisiologia , RNA Viral/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Células-Tronco/enzimologia , Células-Tronco/virologia , Processamento Alternativo , Animais , Encéfalo/enzimologia , Encéfalo/virologia , Linhagem Celular , RNA Helicases DEAD-box/química , Humanos , Imunidade Inata , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Organoides/enzimologia , Organoides/virologia , Infecções por Vírus de RNA/enzimologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Vírus de RNA/genética , Vírus de RNA/imunologia , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Ribonuclease III/química , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Replicação Viral , Zika virus/genética , Zika virus/imunologia , Zika virus/fisiologia , Infecção por Zika virus/enzimologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia
7.
Nano Lett ; 21(15): 6584-6591, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34286581

RESUMO

Lipid nanoparticle SNAs (LNP-SNAs) have been synthesized for the delivery of DNA and RNA to targets in the cytoplasm of cells. Both the composition of the LNP core and surface-presented DNA sequences contribute to LNP-SNA activity. G-rich sequences enhance the activity of LNP-SNAs compared to T-rich sequences. In the LNP core, increased cholesterol content leads to greater activity. Optimized LNP-SNA candidates reduce the siRNA concentration required to silence mRNA by 2 orders of magnitude compared to liposome-based SNAs. In addition, the LNP-SNA architectures alter biodistribution and efficacy profiles in mice. For example, mRNA within LNP-SNAs injected intravenously is primarily expressed in the spleen, while mRNA encapsulated by LNPs (no DNA on the surface) was expressed primarily in the liver with a relatively small amount in the spleen. These data show that the activity and biodistribution of LNP-SNA architectures are different from those of conventional liposomal SNAs and therefore potentially can be used to target tissues.


Assuntos
Lipídeos , Nanopartículas , Animais , DNA/genética , Camundongos , RNA Mensageiro , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Distribuição Tecidual
8.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203338

RESUMO

Diffuse alveolar hemorrhage (DAH) in systemic lupus erythematosus (SLE) is associated with significant mortality, requiring a thorough understanding of its complex mechanisms to develop novel therapeutics for disease control. Activated p53-dependent apoptosis with dysregulated long non-coding RNA (lncRNA) expression is involved in the SLE pathogenesis and correlated with clinical activity. We examined the expression of apoptosis-related p53-dependent lncRNA, including H19, HOTAIR and lincRNA-p21 in SLE-associated DAH patients. Increased lincRNA-p21 levels were detected in circulating mononuclear cells, mainly in CD4+ and CD14+ cells. Higher expression of p53, lincRNA-p21 and cell apoptosis was identified in lung tissues. Lentivirus-based short hairpin RNA (shRNA)-transduced stable transfectants were created for examining the targeting efficacy in lncRNA. Under pristane stimulation, alveolar epithelial cells had increased p53, lincRNA-p21 and downstream Bax levels with elevated apoptotic ratios. After pristane injection, C57/BL6 mice developed DAH with increased pulmonary expression of p53, lincRNA-p21 and cell apoptosis. Intra-pulmonary delivery of shRNA targeting lincRNA-p21 reduced hemorrhage frequencies and improved anemia status through decreasing Bax expression and cell apoptosis. Our findings demonstrate increased p53-dependent lncRNA expression with accelerated cell apoptosis in the lungs of SLE-associated DAH patients, and show the therapeutic potential of targeting intra-pulmonary lncRNA expression in a pristane-induced model of DAH.


Assuntos
RNA Longo não Codificante/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/fisiologia , Modelos Animais de Doenças , Feminino , Hemorragia/genética , Hemorragia/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/microbiologia , Pneumopatias/genética , Pneumopatias/metabolismo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/microbiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/genética
9.
Nat Commun ; 12(1): 4212, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244496

RESUMO

CSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs targeting most germline-expressed genes. Here we show that the two isoforms of CSR-1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Interferência de RNA , Espermatogênese/genética , Animais , Animais Geneticamente Modificados , Arginina/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Éxons/genética , Feminino , Masculino , Metilação , Oogênese/genética , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Processos de Determinação Sexual/genética
10.
Nat Commun ; 12(1): 4584, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321474

RESUMO

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , Antígenos de Diferenciação/genética , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/farmacologia , Antígenos de Diferenciação/metabolismo , Sítios de Ligação , COVID-19/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Interferon beta/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral/efeitos dos fármacos
11.
Nat Commun ; 12(1): 4061, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210982

RESUMO

PIWI proteins use guide piRNAs to repress selfish genomic elements, protecting the genomic integrity of gametes and ensuring the fertility of animal species. Efficient transposon repression depends on amplification of piRNA guides in the ping-pong cycle, which in Drosophila entails tight cooperation between two PIWI proteins, Aub and Ago3. Here we show that post-translational modification, symmetric dimethylarginine (sDMA), of Aub is essential for piRNA biogenesis, transposon silencing and fertility. Methylation is triggered by loading of a piRNA guide into Aub, which exposes its unstructured N-terminal region to the PRMT5 methylosome complex. Thus, sDMA modification is a signal that Aub is loaded with piRNA guide. Amplification of piRNA in the ping-pong cycle requires assembly of a tertiary complex scaffolded by Krimper, which simultaneously binds the N-terminal regions of Aub and Ago3. To promote generation of new piRNA, Krimper uses its two Tudor domains to bind Aub and Ago3 in opposite modification and piRNA-loading states. Our results reveal that post-translational modifications in unstructured regions of PIWI proteins and their binding by Tudor domains that are capable of discriminating between modification states is essential for piRNA biogenesis and silencing.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Proteínas de Transporte/química , Drosophila/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Feminino , Masculino , Metilação , Modelos Moleculares , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/genética , Domínios Proteicos , Proteína-Arginina N-Metiltransferases , RNA Interferente Pequeno/química
12.
Nat Commun ; 12(1): 4498, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301931

RESUMO

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2',3'-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5'-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5'-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production.


Assuntos
Endorribonucleases/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Insetos/genética , RNA Interferente Pequeno/genética , RNA/genética , Animais , Sequência de Bases , Bombyx , Linhagem Celular , Endorribonucleases/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação , Ácidos Fosfatídicos/química , RNA/química , RNA/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA-Seq/métodos , Testículo/metabolismo
13.
Am J Hum Genet ; 108(9): 1669-1691, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34314705

RESUMO

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.


Assuntos
Deficiências do Desenvolvimento/genética , Proteínas de Drosophila/genética , Oftalmopatias Hereditárias/genética , Deficiência Intelectual/genética , Carioferinas/genética , Anormalidades Musculoesqueléticas/genética , beta Carioferinas/genética , Proteína ran de Ligação ao GTP/genética , Alelos , Sequência de Aminoácidos , Animais , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/patologia , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Oftalmopatias Hereditárias/metabolismo , Oftalmopatias Hereditárias/patologia , Feminino , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genoma Humano , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Carioferinas/antagonistas & inibidores , Carioferinas/metabolismo , Masculino , Anormalidades Musculoesqueléticas/metabolismo , Anormalidades Musculoesqueléticas/patologia , Mutação , Neurônios/metabolismo , Neurônios/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sequenciamento Completo do Genoma , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo
14.
Chem Biol Interact ; 346: 109570, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34217686

RESUMO

Verapamil is reported to prevent scar formation. However, whether verapamil is involved in the ureteral stricture scar and the underlying mechanism need further investigation. Fibroblasts were isolated from ureteral scar tissues. TGF-ß1 stimulation was used to induce fibrosis of fibroblasts. Inhibition of CaMK II was achieved by shRNA transfection. CCK-8 was performed to evaluate cell viability. qRT-PCR was applied to determine the level of mRNA while western blotting was used to determine the level of proteins. Immunofluorescence was used to detect the level of vimentin, collagen I and collagen III. Primary fibroblasts was successfully isolated from ureteral scar tissues. TGF-ß1 stimulation was capable to induce collagen production and fibrosis in primary fibroblasts while inhibition of CaMK II attenuate collagen production. Overexpression of wild type CaMK II lead to further increase of collagen production upon TGF-ß1 stimulation while the mutated CaMK II did not exert this promotion. Treatment of verapamil inhibits TGF-ß1 induced collagen production via inhibiting CaMK II. In present study, we revealed a vital role of Verapamil and CaMK II in the formation of ureteral scar. Verapamil inhibited TGF-ß1 induced collagen fiber formation by regulating CaMK II. Our finding might provide new insight into mechanism of prevention and treatment of ureteral scar.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Verapamil/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Cicatriz/tratamento farmacológico , Cicatriz/metabolismo , Cicatriz/patologia , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Mutagênese , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima/efeitos dos fármacos , Verapamil/uso terapêutico , Vimentina/metabolismo
15.
Cell Prolif ; 54(8): e13088, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34240781

RESUMO

OBJECTIVES: Breast cancer-amplified sequence 3 (BCAS3) was initially found to be amplified in human breast cancer (BRCA); however, there has been little consensus on the functions of BCAS3 in breast tumours. MATERIALS AND METHODS: We analysed BCAS3 expression in BRCA using bio-information tools. Affinity purification and mass spectrometry were employed to identify BCAS3-associated proteins. GST pull-down and ubiquitination assays were performed to analyse the interaction mechanism between BCAS3/p53 and CUL4A-RING E3 ubiquitin ligase (CRL4A) complex. BCAS3 was knocked down individually or in combination with p53 in MCF-7 cells to further explore the biological functions of the BCAS3/p53 axis. The clinical values of BCAS3 for BRCA progression were evaluated via semiquantitative immunohistochemistry (IHC) analysis and Cox regression. RESULTS: We reported that the expression level of BCAS3 in BRCA was higher than that in adjacent normal tissues. High BCAS3 expression promoted growth, inhibited apoptosis and conferred chemoresistance in breast cancer cells. Mechanistically, BCAS3 overexpression fostered BRCA cell growth by interacting with the CRL4A complex and promoting ubiquitination and proteasomal degradation of p53. Furthermore, BCAS3 could regulate cell growth, apoptosis and chemoresistance through a p53-mediated mechanism. Clinically, BCAS3 overexpression was significantly correlated with a malignant phenotype. Moreover, higher expression of BCAS3 correlates with shorter overall survival (OS) in BRCA. CONCLUSIONS: The functional characterization of BCAS3 offers new insights into the oncogenic properties and chemotherapy resistance in breast cancer.


Assuntos
Neoplasias da Mama/patologia , Proteínas de Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas Culina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Taxa de Sobrevida , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
16.
Cell Prolif ; 54(8): e13095, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34254370

RESUMO

OBJECTIVES: Scavenger receptor class A, member 3 (Scara3) was involved in adipogenesis. However, the effect of Scara3 on the switch between osteogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs) remains elusive. MATERIALS AND METHODS: The correlations between SCARA3 with the osteogenic-related were analysed based on the GTEx database. The effects of Scara3 on osteogenic or adipogenic differentiation of BMSCs were evaluated by qPCR, Western blot (WB) and cell staining. The mechanisms of Scara3 regulating Foxo1 and autophagy were validated by co-expression analysis, WB and immunofluorescence. In vivo, Scara3 adeno-associated virus was injected into intra-bone marrow of the aged mice and ovariectomized (OVX) mice whose phenotypes were confirmed by micro-CT, calcein double labelling and immunochemistry (HE and OCN staining). RESULTS: SCARA3 was positively correlated with osteogenic-related genes. Scara3 expression gradually decreased during adipogenesis but increased during osteogenesis. Moreover, the deletion of Scara3 favoured adipogenesis over osteogenesis, whereas overexpression of Scara3 significantly enhanced the osteogenesis at the expense of adipogenesis. Mechanistically, Scara3 controlled the cell fate by promoting Foxo1 expression and autophagy flux. In vivo, Scara3 promoted bone formation and reduced bone marrow fat accumulation in OVX mice. In the aged mice, Scara3 overexpression alleviated bone loss as well. CONCLUSIONS: This study suggested that Scara3 regulated the switch between adipocyte and osteoblast differentiation, which represented a potential therapeutic target for bone loss and osteoporosis.


Assuntos
Adipócitos/citologia , Proteína Forkhead Box O1/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Receptores Depuradores Classe A/metabolismo , Adipócitos/metabolismo , Adipogenia , Envelhecimento , Animais , Autofagia , Diferenciação Celular , Células Cultivadas , Feminino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteogênese , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Depuradores Classe A/antagonistas & inibidores , Receptores Depuradores Classe A/genética
17.
Bioconjug Chem ; 32(6): 1078-1093, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34081855

RESUMO

The prevalence of retinal disorders associated with visual impairment and blindness is increasing worldwide, while most of them remain without effective treatment. Pharmacological and molecular therapy development is hampered by the lack of effective drug delivery into the posterior segment of the eye. Among molecular approaches, RNA-interference (RNAi) features strong advantages, yet delivering it to the inner layer of the retina appears extremely challenging. To address this, we developed an original magnetic nanoparticles (MNPs)-based transfection method that allows the efficient delivery of siRNA in all retinal layers of rat adult retinas through magnetic targeting. To establish delivery of RNAi throughout the retina, we have chosen organotypic retinal explants as an ex vivo model and for future high content screening of molecular drugs. Conversely to classic Magnetofection, and similar to conditions in the posterior chamber of the eye, our methods allows attraction of siRNA complexed to MNPs from the culture media into the explant. Our method termed "Reverse Magnetofection" provides a novel and nontoxic strategy for RNAi-based molecular as well as gene therapy in the retina that can be transferred to a wide variety of organ explants.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Fenômenos Magnéticos , RNA Interferente Pequeno/metabolismo , Retina/citologia , Animais , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Transfecção
18.
Molecules ; 26(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072265

RESUMO

Though siRNA-based therapy has achieved great progress, efficient siRNA delivery remains a challenge. Here, we synthesized a copolymer PAsp(-N=C-PEG)-PCys-PAsp(DETA) consisting of a poly(aspartate) block grafted with comb-like PEG side chains via a pH-sensitive imine bond (PAsp(-N=C-PEG) block), a poly(l-cysteine) block with a thiol group (PCys block), and a cationic poly(aspartate) block grafted with diethylenetriamine (PAsp(DETA) block). The cationic polymers efficiently complexed siRNA into polyplexes, showing a sandwich-like structure with a PAsp(-N=C-PEG) out-layer, a crosslinked PCys interlayer, and a complexing core of siRNA and PAsp(DETA). Low pH-triggered breakage of pH-sensitive imine bonds caused PEG shedding. The disulfide bond-crosslinking and pH-triggered PEG shedding synergistically decreased the polyplexes' size from 75 nm to 26 nm. To neutralize excessive positive charges and introduce the targeting ligand, the polyplexes without a PEG layer were coated with an anionic copolymer modified with the targeting ligand lauric acid. The resulting polyplexes exhibited high transfection efficiency and lysosomal escape capacity. This study provides a promising strategy to engineer the size and surface of polyplexes, allowing long blood circulation and targeted delivery of siRNA.


Assuntos
Polímeros/química , RNA Interferente Pequeno/metabolismo , Ânions , Cátions , Sobrevivência Celular , Reagentes para Ligações Cruzadas/química , Citoplasma/metabolismo , Dissulfetos , Sistemas de Liberação de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Ácidos Láuricos/química , Ligantes , Espectroscopia de Ressonância Magnética , Oxigênio/química , Tamanho da Partícula , Polietilenoglicóis/química , Espectroscopia de Infravermelho com Transformada de Fourier , Células THP-1
19.
Nat Commun ; 12(1): 3444, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103528

RESUMO

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function.


Assuntos
Arginina/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Antineoplásicos/farmacologia , Biocatálise/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Metilação/efeitos dos fármacos , Camundongos Nus , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína-Arginina N-Metiltransferases/deficiência , Proteínas Proto-Oncogênicas c-akt/química , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos
20.
Nat Commun ; 12(1): 3492, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108460

RESUMO

In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with translating ribosomes, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Uso do Códon , Biossíntese de Proteínas , RNA Interferente Pequeno/biossíntese , Animais , Proteínas Argonauta/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Catálise , Citosol/metabolismo , Mutação , Oogônios/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Ribossomos/metabolismo
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