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1.
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
2.
Nat Commun ; 12(1): 4126, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226554

RESUMO

Double stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.


Assuntos
Proteína BRCA1/metabolismo , RNA Helicases DEAD-box/metabolismo , DNA , Recombinação Homóloga , Proteínas de Neoplasias/metabolismo , RNA , Proteína BRCA1/genética , RNA Helicases DEAD-box/genética , Dano ao DNA , DNA Helicases , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Ftalazinas , Piperazinas , RNA Helicases , RNA Mensageiro , Rad51 Recombinase , Reparo de DNA por Recombinação
3.
Eur J Med Res ; 26(1): 75, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34256840

RESUMO

BACKGROUND: The aim of this study was to evaluate the expression of four up/down-regulated inflammatory miRNAs and their mRNA targets in the serum samples of COVID-19 patients with different grades. Also, we investigated the relative expression of these miRNAs and mRNAs during hospitalization. METHODS: In this cross-sectional study, 5 mL of blood sample were taken from COVID-19 patients with different grades and during hospitalization from several health centers of Yazd, Tehran, and Zahedan province of Iran from December 20, 2020 to March 2, 2021. The relative expression of miRNAs and mRNAs was evaluated by q-PCR. RESULTS: We found that the relative expression of hsa-miR-31-3p, hsa-miR-29a-3p, and hsa-miR-126-3p was significantly decreased and the relative expression of their mRNA targets (ZMYM5, COL5A3, and CAMSAP1) was significantly increased with the increase of disease grade. Conversely, the relative expression of hsa-miR-17-3p was significantly increased and its mRNA target (DICER1) was significantly decreased with the increase of disease grade. This pattern was exactly seen during hospitalization of COVID-19 patients who did not respond to treatment. In COVID-19 patients who responded to treatment, the expression of selected miRNAs and their mRNA targets returned to the normal level. A negative significant correlation was seen between (1) the expression of hsa-miR-31-3p and ZMYM5, (2) hsa-miR-29a-3p and COL5A3, (3) hsa-miR-126-3p and CAMSAP1, and (4) hsa-miR-17-3p and DICER1 in COVID-19 patients with any grade (P < 0.05) and during hospitalization. CONCLUSIONS: In this study, we gained a more accurate understanding of the expression of up/down-regulated inflammatory miRNAs in the blood of COVID-19 patients. The obtained data may help us in the diagnosis and prognosis of COVID-19. TRIAL REGISTRATION: The ethics committee of Zahedan University of Medical Sciences, Zahedan, Iran. (Ethical Code: IR.ZAUMS.REC.1399.316) was registered for this project.


Assuntos
COVID-19/genética , Perfilação da Expressão Gênica , MicroRNAs/genética , RNA Mensageiro/genética , COVID-19/sangue , COVID-19/virologia , Proteínas de Transporte/genética , Colágeno/genética , Estudos Transversais , RNA Helicases DEAD-box/genética , Hospitalização/estatística & dados numéricos , Humanos , Irã (Geográfico) , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Ribonuclease III/genética , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença
4.
Nat Commun ; 12(1): 3397, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099665

RESUMO

It is known that an RNA's structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact (i.e., full length) RNA structures will facilitate investigations of the functions and regulation mechanisms of small RNAs and identify short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo selective 2'-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer based on the combination of RNA immunoprecipitation pull-down and icSHAPE-MaP small RNA structural profiling. We discover distinct structural categories of Dicer substrates in correlation to both their binding affinity and cleavage efficiency. And by tertiary structural modeling constrained by icSHAPE-MaP RNA structural data, we find the spatial distance measuring as an influential parameter for Dicer cleavage-site selection.


Assuntos
RNA Helicases DEAD-box/metabolismo , Conformação de Ácido Nucleico , RNA/química , Ribonuclease III/metabolismo , Biologia Computacional , RNA Helicases DEAD-box/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , RNA/genética , RNA/metabolismo , Sondas RNA , RNA-Seq , Ribonuclease III/genética , Especificidade por Substrato/genética
5.
Aging (Albany NY) ; 13(9): 12766-12779, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33952717

RESUMO

Pain in hepatocellular carcinoma (HCC) is a frequent cause of low quality of life, and morphine is routinely used as a first-line opiate analgesic in HCC. Morphine may exert not only analgesic effects but also anti-cancer effects via unknown mechanisms. Here we show that morphine can inhibit HCC cell proliferation. We further show that DEAD-box helicase 49 (DDX49) is up-regulated in HCC tumors, and that knocking down the DDX49 gene decreases tumor formation in vivo and in vitro, as well as reduces tumor metastasis in vivo. Morphine decreases DDX49 expression in HCC cells. Our results suggest that DDX49 contributes to HCC, and that morphine may exert anti-cancer effects by down-regulating it.


Assuntos
Dor do Câncer/tratamento farmacológico , Carcinoma Hepatocelular/terapia , RNA Helicases DEAD-box/genética , Neoplasias Hepáticas/terapia , Morfina/farmacologia , Idoso , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Dor do Câncer/etiologia , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimioterapia Adjuvante/métodos , RNA Helicases DEAD-box/antagonistas & inibidores , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Hepatectomia , Humanos , Fígado/patologia , Fígado/cirurgia , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Morfina/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Nat Commun ; 12(1): 3082, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035302

RESUMO

Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure. Here we show that Prp28, a yeast DEAD-box ATPase, transiently interacts with the conserved 5' splice-site (5'SS) GU dinucleotide and makes splicing-dependent contacts with the U1 snRNP protein U1C, and U4/U6.U5 tri-snRNP proteins, Prp8, Brr2, and Snu114. We further show that Prp28's ATPase activity is potentiated by the phosphorylated Npl3, but not the unphosphorylated Npl3, thus suggesting a strategy for regulating DExD/H-box ATPases. We propose that Npl3 is a functional counterpart of the metazoan-specific Prp28 N-terminal region, which can be phosphorylated and serves as an anchor to human spliceosome.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas Nucleares/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , RNA Helicases/genética , RNA Helicases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Ribonuclease H/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética
7.
Nucleic Acids Res ; 49(8): 4456-4471, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33823555

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) expresses miRNAs during latency. However, regulation of viral miRNAs remains largely unknown. Our prior studies demonstrated that MCPIP1 regulates KSHV miRNA biogenesis by degrading most KSHV pre-miRNAs through its RNase activity. Some viral pre-miRNAs are partially resistant to degradation by MCPIP1. Here, we further characterized MCPIP1 substrate specificity and its antiviral potential against KSHV infection. In vitro cleavage assays and binding assays showed that MCPIP1 cleavage efficiency is related to binding affinity. Motif-based sequence analysis identified that KSHV pre-miRNAs that are well degraded by MCPIP1 have a 5-base motif (M5 base motif) within their terminal loops and this motif region consists of multiple pyrimidine-purine-pyrimidine (YRY) motifs. We further demonstrated that mutation of this M5 base motif within terminal loop of pre-miRNAs inhibited MCPIP1-mediated RNA degradation. We also revealed that MCPIP1 has an antiviral effect against KSHV infection. MCPIP1 can reduce the expression of Dicer, which in turn restricts KSHV infection. Conclusively, our findings demonstrated that MCPIP1 inhibited KSHV infection and suppressed viral miRNA biogenesis by directly degrading KSHV pre-miRNAs and altering the expression of miRNA biogenesis factors.


Assuntos
Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 8/metabolismo , MicroRNAs/metabolismo , RNA Viral/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Técnicas de Silenciamento de Genes , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/genética , Humanos , MicroRNAs/genética , Motivos de Nucleotídeos , Ligação Proteica , Estabilidade de RNA/genética , RNA Viral/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo
8.
Aging (Albany NY) ; 13(7): 10387-10395, 2021 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-33819916

RESUMO

Emerging studies have noted that dysregulated lncRNAs are implicated in cancer progression and tumorigenesis. We first showed that MSC-AS1 was overexpressed in gastric cancer (GC) cells (HGC-27, MKN-45, SGC-7901 and MGC-803 cells) compared with GES cells. We observed that MSC-AS1 was upregulated in GC specimens compared with paired normal specimens. MSC-AS1 increased cell growth and cycle progression. Moreover, the overexpression of MSC-AS1 enhanced the secretion of the inflammatory mediators IL-1ß, IL-6 and TNF-α. We found that the overexpression of MSC-AS1 inhibited the expression of miR-142-5p in HGC-27 cells. We noted that DDK5 was a target gene of miR-142-5p. The overexpression of miR-142-5p suppressed the luciferase activity of wild-type DDX5, but the luciferase activity of the mutant DDX5 was not changed. We showed that miR-142-5p was downregulated in GC specimens compared with paired normal specimens. MSC-AS1 expression was inversely correlated with miR-142-5p expression in GC specimens. MSC-AS1 induced cell growth, cell cycle progression and inflammatory mediator secretion by modulating DDX5. These results showed that MSC-AS1 functions as a key oncogene in the development of GC.


Assuntos
Carcinoma/patologia , RNA Helicases DEAD-box/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/patologia , Carcinoma/genética , Carcinoma/metabolismo , Proliferação de Células , RNA Helicases DEAD-box/genética , Humanos , Mediadores da Inflamação/metabolismo , MicroRNAs/genética , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo
9.
Nucleic Acids Res ; 49(9): 5336-5350, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33905506

RESUMO

DDX3 is an RNA chaperone of the DEAD-box family that regulates translation. Ded1, the yeast ortholog of DDX3, is a global regulator of translation, whereas DDX3 is thought to preferentially affect a subset of mRNAs. However, the set of mRNAs that are regulated by DDX3 are unknown, along with the relationship between DDX3 binding and activity. Here, we use ribosome profiling, RNA-seq, and PAR-CLIP to define the set of mRNAs that are regulated by DDX3 in human cells. We find that while DDX3 binds highly expressed mRNAs, depletion of DDX3 particularly affects the translation of a small subset of the transcriptome. We further find that DDX3 binds a site on helix 16 of the human ribosomal rRNA, placing it immediately adjacent to the mRNA entry channel. Translation changes caused by depleting DDX3 levels or expressing an inactive point mutation are different, consistent with different association of these genetic variant types with disease. Taken together, this work defines the subset of the transcriptome that is responsive to DDX3 inhibition, with relevance for basic biology and disease states where DDX3 is altered.


Assuntos
Regiões 5' não Traduzidas , RNA Helicases DEAD-box/fisiologia , Biossíntese de Proteínas , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Humanos , Mutação , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , RNA Interferente Pequeno
10.
Methods Mol Biol ; 2300: 133-139, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33792878

RESUMO

MicroRNAs (miRNAs) are a class of small noncoding single-stranded RNA molecules containing 18-22 nucleotides that play an important role in the regulation of gene expression at the post-transcriptional and translational levels. Loss-of-function studies are the fundamental strategy to examine miRNA function and target genes in cellular and molecular biology. Traditional methods for miRNA loss-of-function studies include miRNA-specific antisense inhibitors, miRNA sponges, and genetic knockout. However, efficiency, specificity, and stability of these methods are not adequate. Our study suggests that CRISPR/Cas9 is an economic, convenient, and innovative strategy with high efficiency, specificity, and stability for the modulation of miRNA expression. Herein, we describe a detailed protocol for knocking out miRNA genes in vitro and in vivo with the CRISPR/Cas9 system.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Silenciamento de Genes/métodos , MicroRNAs/genética , RNA Mensageiro/genética , Animais , RNA Helicases DEAD-box/genética , Edição de Genes , Regulação da Expressão Gênica , Camundongos , Plasmídeos/genética , Ribonuclease III/genética , Transfecção
11.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804185

RESUMO

The progress of the cell cycle is directly regulated by modulation of cyclins and cyclin-dependent kinases. However, many proteins that control DNA replication, RNA transcription and the synthesis and degradation of proteins can manage the activity or levels of master cell cycle regulators. Among them, RNA helicases are key participants in RNA metabolism involved in the global or specific tuning of cell cycle regulators at the level of transcription and translation. Several RNA helicases have been recently evaluated as promising therapeutic targets, including eIF4A, DDX3 and DDX5. However, targeting RNA helicases can result in side effects due to the influence on the cell cycle. In this review, we discuss direct and indirect participation of RNA helicases in the regulation of the cell cycle in order to draw attention to downstream events that may occur after suppression or inhibition of RNA helicases.


Assuntos
Ciclo Celular/genética , Replicação do DNA/genética , RNA Helicases/genética , Divisão Celular/genética , Quinases Ciclina-Dependentes/genética , RNA Helicases DEAD-box/genética , Fator de Iniciação 4A em Eucariotos/genética , Humanos
12.
Theranostics ; 11(9): 4516-4530, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33754075

RESUMO

Rationale: Accumulating evidence indicates that long noncoding RNAs (lncRNAs) play crucial roles in cancer progression; however, only few have been characterized in detail. The current study aimed to identify a novel cancer driver lncRNA in glioblastoma and colon adenocarcinoma. Methods: We performed whole transcriptome analysis of TCGA pan-cancer datasets to compare the lncRNA expression profiles of tumor and paired normal tissues. In situ hybridization of tissue sections was performed to validate the expression data and determine the localization of lncRNAs that may be linked to glioblastoma and colon adenocarcinoma. Chromatin isolation by RNA purification (ChIRP), chromatin immunoprecipitation (ChIP), and Co-immunoprecipitation (Co-IP) assays were performed to assess the interaction between lncRNA, proteins, and chromatin. The functional significance of the identified lncRNAs was verified in vitro and in vivo by knockdown or exogenous expression experiments. Results: We found a lncRNA ENST00000449248.1 termed PRC2 and DDX5 associated lncRNA (PRADX) that is highly expressed in glioblastoma and colon adenocarcinoma cells and tissues. PRADX, mainly located in the nucleus of tumor cells, could bind to EZH2 protein via the 5' terminal sequence. Moreover, PRADX increased the trimethylation of H3K27 in the UBXN1 gene promoter via PRC2/DDX5 complex recruitment and promoted NF-κB activity through UBXN1 suppression. Knockdown of PRADX significantly inhibited tumor cell viability and clonogenic growth in vitro. In xenograft models, PRADX knockdown suppressed tumor growth and tumorigenesis and prolonged the survival of tumor-bearing mice. Conclusions: PRADX acts as a cancer driver and may serve as a potential therapeutic target for glioblastoma and colon adenocarcinoma.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinogênese/genética , RNA Helicases DEAD-box/genética , NF-kappa B/genética , RNA Longo não Codificante/genética , Adenocarcinoma/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Cromatina/genética , Imunoprecipitação da Cromatina/métodos , Neoplasias do Colo/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/genética , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética
13.
Acta Biochim Biophys Sin (Shanghai) ; 53(4): 463-471, 2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33751023

RESUMO

A large number of proteins involved in RNA metabolism possess a double-stranded RNA-binding domain (dsRBD), whose sequence variations and functional versatilities are still being recognized. All dsRBDs have a similar structural fold: α1-L1-ß1-L2-ß2-L3-ß3-L4-α2 (α represents an α-helix, ß a ß-sheet, and L a loop conformation between the well-defined secondary structures). Our recent work revealed that the dsRBD in Drosha, which is involved in animal microRNA (miRNA) biogenesis, differs from other dsRBDs by containing a short insertion in its L1 region and that this insertion is important for Drosha function. We asked why the same insertion is excluded in all other dsRBDs and proposed that a longer L1 may be detrimental to their functions. In this study, to test this hypothesis, we inserted the Drosha sequence into several well-known dsRBDs from various organisms. Gel mobility shift assay demonstrated that L1 extension invariably reduced RNA binding by these dsRBDs. In addition, such a mutation in Dicer, another protein involved in miRNA biogenesis, impaired Dicer's ability to process miRNAs, which led to de-repression of reporter expression, in human cells. Taken together, our results add to the growing appreciation of the diversity in dsRBDs and suggest that dsRBDs have intricate structures and functions that are sensitive to perturbations in the L1 region.


Assuntos
Motivo de Ligação ao RNA de Cadeia Dupla , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Animais , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/fisiologia , DNA de Cadeia Simples/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Células HEK293 , Humanos , MicroRNAs/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Estrutura Secundária de Proteína , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Ribonuclease III/química , Ribonuclease III/genética , Ribonuclease III/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/fisiologia
14.
Urologe A ; 60(6): 776-779, 2021 Jun.
Artigo em Alemão | MEDLINE | ID: mdl-33728475

RESUMO

Cystic nephroma is a rare tumor in childhood. Seventy percent of all cases are associated with DICER1-anomaly and therefore cystic nephroma represents the second most common tumor of all patients with DICER1 mutation. We present a case of a 15-month-old boy with a DICER1 mutation and cystic nephroma. Organ-sparing surgery was not possible. Due to higher prevalence of germ cell tumors, follow-up after nephrectomy is necessary until adulthood.


Assuntos
Neoplasias Renais , Segunda Neoplasia Primária , Adulto , RNA Helicases DEAD-box/genética , Humanos , Lactente , Neoplasias Renais/diagnóstico por imagem , Neoplasias Renais/genética , Neoplasias Renais/cirurgia , Masculino , Mutação , Nefrectomia , Ribonuclease III/genética
15.
Cell Mol Life Sci ; 78(7): 3709-3724, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33733306

RESUMO

Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform-PAZ-Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.


Assuntos
RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Quadruplex G , MicroRNAs/metabolismo , RNA/metabolismo , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , RNA Helicases DEAD-box/metabolismo , DNA/química , DNA/genética , Inibidores Enzimáticos/química , Humanos , MicroRNAs/genética , Conformação de Ácido Nucleico , Conformação Proteica , RNA/química , RNA/genética , Ribonuclease III/metabolismo
16.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669056

RESUMO

Warsaw breakage syndrome (WABS) is a genetic disorder characterized by sister chromatid cohesion defects, growth retardation, microcephaly, hearing loss and other variable clinical manifestations. WABS is due to biallelic mutations of the gene coding for the super-family 2 DNA helicase DDX11/ChlR1, orthologous to the yeast chromosome loss protein 1 (Chl1). WABS is classified in the group of "cohesinopathies", rare hereditary diseases that are caused by mutations in genes coding for subunits of the cohesin complex or protein factors having regulatory roles in the sister chromatid cohesion process. In fact, among the cohesion regulators, an important player is DDX11, which is believed to be important for the functional coupling of DNA synthesis and cohesion establishment at the replication forks. Here, we will review what is known about the molecular and cellular functions of human DDX11 and its role in WABS etiopathogenesis, even in light of recent findings on the role of cohesin and its regulator network in promoting chromatin loop formation and regulating chromatin spatial organization.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , RNA Helicases DEAD-box/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Doenças Raras/metabolismo , Anormalidades Múltiplas/genética , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Cromátides/patologia , Cromatina/patologia , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , RNA Helicases DEAD-box/genética , Replicação do DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Mutação , Filogenia , Doenças Raras/congênito , Doenças Raras/enzimologia , Doenças Raras/fisiopatologia
17.
Cell Prolif ; 54(5): e13000, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33666296

RESUMO

OBJECTIVES: Mammalian spermatogenesis is a biological process of male gamete formation. Gonocytes are the only precursors of spermatogonial stem cells (SSCs) which develop into mature spermatozoa. DDX5 is one of DEAD-box RNA helicases and expresses in male germ cells, suggesting that Ddx5 plays important functions during spermatogenesis. Here, we explore the functions of Ddx5 in regulating the specification of gonocytes. MATERIALS AND METHODS: Germ cell-specific Ddx5 knockout (Ddx5-/- ) mice were generated. The morphology of testes and epididymides and fertility in both wild-type and Ddx5-/- mice were analysed. Single-cell RNA sequencing (scRNA-seq) was used to profile the transcriptome in testes from wild-type and Ddx5-/- mice at postnatal day (P) 2. Dysregulated genes were validated by single-cell qRT-PCR and immunofluorescent staining. RESULTS: In male mice, Ddx5 was expressed in germ cells at different stages of development. Germ cell-specific Ddx5 knockout adult male mice were sterile due to completely devoid of germ cells. Male germ cells gradually disappeared in Ddx5-/- mice from E18.5 to P6. Single-cell transcriptome analysis showed that genes involved in cell cycle and glial cell line-derived neurotrophic factor (GDNF) pathway were significantly decreased in Ddx5-deficient gonocytes. Notably, Ddx5 ablation impeded the proliferation of gonocytes. CONCLUSIONS: Our study reveals the critical roles of Ddx5 in fate determination of gonocytes, offering a novel insight into the pathogenesis of male sterility.


Assuntos
RNA Helicases DEAD-box/metabolismo , Células Germinativas/metabolismo , Animais , Animais Recém-Nascidos , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Células Germinativas/citologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Infertilidade/metabolismo , Infertilidade/patologia , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência de RNA , Análise de Célula Única , Testículo/metabolismo , Testículo/patologia
18.
BMC Bioinformatics ; 22(1): 63, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568063

RESUMO

BACKGROUND: Human dicer is an enzyme that cleaves pre-miRNAs into miRNAs. Several models have been developed to predict human dicer cleavage sites, including PHDCleav and LBSizeCleav. Given an input sequence, these models can predict whether the sequence contains a cleavage site. However, these models only consider each sequence independently and lack interpretability. Therefore, it is necessary to develop an accurate and explainable predictor, which employs relations between different sequences, to enhance the understanding of the mechanism by which human dicer cleaves pre-miRNA. RESULTS: In this study, we develop an accurate and explainable predictor for human dicer cleavage site - ReCGBM. We design relational features and class features as inputs to a lightGBM model. Computational experiments show that ReCGBM achieves the best performance compared to the existing methods. Further, we find that features in close proximity to the center of pre-miRNA are more important and make a significant contribution to the performance improvement of the developed method. CONCLUSIONS: The results of this study show that ReCGBM is an interpretable and accurate predictor. Besides, the analyses of feature importance show that it might be of particular interest to consider more informative features close to the center of the pre-miRNA in future predictors.


Assuntos
RNA Helicases DEAD-box , MicroRNAs , Precursores de RNA , Ribonuclease III , RNA Helicases DEAD-box/genética , Humanos , MicroRNAs/genética , Clivagem do RNA , Ribonuclease III/genética
19.
Methods Mol Biol ; 2218: 61-73, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606223

RESUMO

Primordial germ cells (PGCs) are the precursor cells that form during early embryogenesis and later differentiate into oocytes or spermatozoa. Abnormal development of PGCs is frequently a causative factor of infertility and germ cell tumors. However, our understanding of PGC development remains insufficient, and we have few pharmacological tools for manipulating PGC development for biological study or therapy. The zebrafish (Danio rerio) embryos provide an excellent in vivo animal model to study PGCs, because zebrafish embryos are transparent and develop outside the mother. Importantly, the model is also amenable to facile chemical manipulations, including scalable screening to discover novel compounds that alter PGC development. This chapter describes methodologies for manipulating the germline (i.e., PGCs) with small molecules and for monitoring PGC development. Utilizing the 3'UTR of PGC marker genes such as nanos3 and ddx4/vasa is a key component of these methodologies, which consist of expressing fluorescent or luminescent proteins in PGCs, treatment with small molecules, and quantitative observation of PGC development.


Assuntos
Células Germinativas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Regiões 3' não Traduzidas/genética , Animais , RNA Helicases DEAD-box/genética , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Feminino , Proteínas Luminescentes/genética , Masculino , Proteínas de Ligação a RNA/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
20.
Genes (Basel) ; 12(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33535521

RESUMO

DEAD-box RNA helicases are ubiquitous proteins found in all kingdoms of life and that are associated with all processes involving RNA. Their central roles in biology make these proteins potential targets for therapeutic or prophylactic drugs. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest because of their important role(s) in translation. In this paper, we identified and aligned the protein sequences of 28 different DEAD-box proteins from the kinetoplast-protozoan parasite Leishmania infantum, which is the cause of the visceral form of leishmaniasis that is often lethal if left untreated, and compared them with the consensus sequence derived from DEAD-box proteins in general, and from the Ded1/DDX3 subfamily in particular, from a wide variety of other organisms. We identified three potential homologs of the Ded1/DDX3 subfamily and the equivalent proteins from the related protozoan parasite Trypanosoma brucei, which is the causative agent of sleeping sickness. We subsequently tested these proteins for their ability to complement a yeast strain deleted for the essential DED1 gene. We found that the DEAD-box proteins from Trypanosomatids are highly divergent from other eukaryotes, and consequently they are suitable targets for protein-specific drugs.


Assuntos
RNA Helicases DEAD-box/genética , Proteínas de Saccharomyces cerevisiae/genética , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/genética , Sequência de Aminoácidos/genética , Simulação por Computador , Humanos , Leishmania infantum/genética , Leishmania infantum/patogenicidade , Biossíntese de Proteínas/genética , RNA/genética , Saccharomyces cerevisiae/genética , Trypanosoma brucei brucei/patogenicidade , Tripanossomíase Africana/parasitologia
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