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1.
JAMA Netw Open ; 4(2): e210112, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33630087

RESUMO

Importance: Genetic disorders are historically defined through phenotype-first approaches. However, risk estimates derived from phenotype-linked ascertainment may overestimate severity and penetrance. Pathogenic variants in DICER1 are associated with increased risks of rare and common neoplasms and thyroid disease in adults and children. This study explored how effectively a genome-first approach could characterize the clinical traits associated with germline DICER1 putative loss-of-function (pLOF) variants in an unselected clinical cohort. Objective: To examine the prevalence, penetrance, and phenotypic characteristics of carriers of germline DICER1 pLOF variants via genome-first ascertainment. Design, Setting, and Participants: This cohort study classifies DICER1 variants in germline exome sequence data from 92 296 participants of the Geisinger MyCode Community Health Initiative. Data for each MyCode participant were used from the start of the Geisinger electronic health record to February 1, 2018. Main Outcomes and Measures: Prevalence of germline DICER1 variation; penetrance of malignant tumors and thyroid disease in carriers of germline DICER1 variation; structured, manual review of electronic health records; and DICER1 sequencing of available tumors from an associated cancer registry. Results: A total of 92 296 adults (mean [SD] age, 59 [18] years; 98% white; 60% female) participated in the study. Germline DICER1 pLOF variants were observed in 1 in 3700 to 1 in 4600 participants, more than double the expected prevalence. Malignant tumors (primarily thyroid carcinoma) were observed in 4 of 25 participants (16%) with DICER1 pLOF variants, which is comparable (by 50 years of age) to the frequency of neoplasms in the largest registry- and clinic-based (phenotype-first) DICER1 studies published to date. DICER1 pLOF variants were significantly associated with risks of thyroidectomy (odds ratio [OR], 6.0; 95% CI, 2.2-16.3; P = .007) and thyroid cancer (OR, 9.2; 95% CI, 2.1-34.7; P = .02) compared with controls, but there was not a significant increase in the risk of goiter (OR, 1.8; 95% CI, 0.7-4.9). A female patient in her 80s who was a carrier of a germline DICER1 hotspot variant was apparently healthy on electronic health record review. The term DICER1 did not appear in any of the medical records of the 25 participants with a pLOF DICER1 variant, even in those affected with a known DICER1-associated tumor or thyroid phenotype. Conclusions and Relevance: This cohort study was able to ascertain individuals with germline DICER1 variants based on a genome-first approach rather than through a previously established DICER1-related phenotype. Use of the genome-first approach may complement more traditional approaches to syndrome delineation and may be an efficient approach for risk estimation.


Assuntos
RNA Helicases DEAD-box/genética , Penetrância , Fenótipo , Ribonuclease III/genética , Doenças da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Genoma , Mutação em Linhagem Germinativa , Bócio Nodular/epidemiologia , Bócio Nodular/genética , Doença de Graves/epidemiologia , Doença de Graves/genética , Heterozigoto , Humanos , Hipotireoidismo/epidemiologia , Hipotireoidismo/genética , Neoplasias Renais/epidemiologia , Neoplasias Renais/genética , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Neoplasias/epidemiologia , Neoplasias/genética , Neoplasias Ovarianas/epidemiologia , Neoplasias Ovarianas/genética , Prevalência , Blastoma Pulmonar/epidemiologia , Blastoma Pulmonar/genética , Sarcoma/epidemiologia , Sarcoma/genética , Tumor de Células de Sertoli-Leydig/epidemiologia , Tumor de Células de Sertoli-Leydig/genética , Tumores do Estroma Gonadal e dos Cordões Sexuais/epidemiologia , Tumores do Estroma Gonadal e dos Cordões Sexuais/genética , Neoplasias Testiculares/epidemiologia , Neoplasias Testiculares/genética , Doenças da Glândula Tireoide/epidemiologia , Neoplasias da Glândula Tireoide/epidemiologia , Nódulo da Glândula Tireoide/epidemiologia , Nódulo da Glândula Tireoide/genética , Tireoidectomia/estatística & dados numéricos , Tireotoxicose/epidemiologia , Tireotoxicose/genética , Tumor de Wilms/epidemiologia , Tumor de Wilms/genética , Adulto Jovem
2.
Microbiome ; 9(1): 57, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637135

RESUMO

BACKGROUND: Plants are naturally associated with root microbiota, which are microbial communities influential to host fitness. Thus, it is important to understand how plants control root microbiota. Epigenetic factors regulate the readouts of genetic information and consequently many essential biological processes. However, it has been elusive whether RNA-directed DNA methylation (RdDM) affects root microbiota assembly. RESULTS: By applying 16S rRNA gene sequencing, we investigated root microbiota of Arabidopsis mutants defective in the canonical RdDM pathway, including dcl234 that harbors triple mutation in the Dicer-like proteins DCL3, DCL2, and DCL4, which produce small RNAs for RdDM. Alpha diversity analysis showed reductions in microbe richness from the soil to roots, reflecting the selectivity of plants on root-associated bacteria. The dcl234 triple mutation significantly decreases the levels of Aeromonadaceae and Pseudomonadaceae, while it increases the abundance of many other bacteria families in the root microbiota. However, mutants of the other examined key players in the canonical RdDM pathway showed similar microbiota as Col-0, indicating that the DCL proteins affect root microbiota in an RdDM-independent manner. Subsequently gene analysis by shotgun sequencing of root microbiome indicated a selective pressure on microbial resistance to plant defense in the dcl234 mutant. Consistent with the altered plant-microbe interactions, dcl234 displayed altered characters, including the mRNA and sRNA transcriptomes that jointly highlighted altered cell wall organization and up-regulated defense, the decreased cellulose and callose deposition in root xylem, and the restructured profile of root exudates that supported the alterations in gene expression and cell wall modifications. CONCLUSION: Our findings demonstrate an important role of the DCL proteins in influencing root microbiota through integrated regulation of plant defense, cell wall compositions, and root exudates. Our results also demonstrate that the canonical RdDM is dispensable for Arabidopsis root microbiota. These findings not only establish a connection between root microbiota and plant epigenetic factors but also highlight the complexity of plant regulation of root microbiota. Video abstract.


Assuntos
Arabidopsis/metabolismo , Arabidopsis/microbiologia , Metilação de DNA/genética , Microbiota , Raízes de Plantas/microbiologia , RNA de Plantas , Ribonuclease III/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Microbiota/genética , Raízes de Plantas/genética , RNA Ribossômico 16S/genética , Ribonuclease III/genética
3.
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
4.
Nucleic Acids Res ; 49(2): 601-620, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33406242

RESUMO

It is a well-known and intensively studied phenomenon that the levels of many miRNAs are differentiated in cancer. miRNA biogenesis and functional expression are complex processes orchestrated by many proteins cumulatively called miRNA biogenesis proteins. To characterize cancer somatic mutations in the miRNA biogenesis genes and investigate their potential impact on the levels of miRNAs, we analyzed whole-exome sequencing datasets of over 10 000 cancer/normal sample pairs deposited within the TCGA repository. We identified and characterized over 3600 somatic mutations in 29 miRNA biogenesis genes and showed that some of the genes are overmutated in specific cancers and/or have recurrent hotspot mutations (e.g. SMAD4 in PAAD, COAD and READ; DICER1 in UCEC; PRKRA in OV and LIN28B in SKCM). We identified a list of miRNAs whose level is affected by particular types of mutations in either SMAD4, SMAD2 or DICER1 and showed that hotspot mutations in the RNase domains in DICER1 not only decrease the level of 5p-miRNAs but also increase the level of 3p-miRNAs, including many well-known cancer-related miRNAs. We also showed an association of the mutations with patient survival. Eventually, we created an atlas/compendium of miRNA biogenesis alterations providing a useful resource for different aspects of biomedical research.


Assuntos
RNA Helicases DEAD-box/genética , MicroRNAs/biossíntese , Mutação , Proteínas de Neoplasias/genética , Neoplasias/genética , RNA Neoplásico/biossíntese , Ribonuclease III/genética , Proteína Smad2/genética , Proteína Smad4/genética , RNA Helicases DEAD-box/metabolismo , Conjuntos de Dados como Assunto , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Estudo de Associação Genômica Ampla , Humanos , MicroRNAs/genética , Modelos Moleculares , Mutação de Sentido Incorreto , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/mortalidade , Conformação Proteica , RNA Neoplásico/genética , Ribonuclease III/metabolismo , Proteína Smad2/química , Proteína Smad2/metabolismo , Proteína Smad4/química , Proteína Smad4/metabolismo
5.
Gene ; 766: 145132, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32911029

RESUMO

Dysfunctions in mechanisms of gene regulation based on RNA interference are recognized as a common feature of the molecular basis of cancer pathogenesis. Therefore, as one of the crucial components of the machinery involved in the biogenesis of both siRNAs and microRNA molecules, DICER was recognized as one of the candidates for the research in the field of carcinogenesis. Due to their potential functional properties, several genetic variants located within DICER1 gene were analyzed for their possible association with the susceptibility to cancer through case-control studies. In order to elucidate their effect on the overall cancer risk, we conducted an updated meta-analysis of all eligible association studies. The publications were selected based on PubMed database search, while OpenMeta-analyst and MetaGenyo software were used for quantitative data synthesis. Statistically significant results were found for the association of rs1057035 with the overall cancer risk under multiple genetic models (PCT vs. TT < 0.001, ORCT vs. TT = 0.870, 95% CI = 0.812-0.933; Pallelic = 0.009, ORallelic = 0.896, 95% CI = 0.825-0.973; Pdom < 0.001, ORdom = 0.874, 95% CI = 0.817-0.934; Poverdom = 0.004, ORoverdom = 0.858, 95% CI = 0.773-0.953). Other selected genetic variants within DICER1, rs13078, rs1209904 and rs3742330, did not show the association with the overall susceptibility to malignant diseases. We conclude that rs1057035 may represent a potential biomarker associated with the risk of developing cancer, which requires a confirmation in a larger set of studies.


Assuntos
RNA Helicases DEAD-box/genética , Predisposição Genética para Doença/genética , Neoplasias/genética , Polimorfismo de Nucleotídeo Único/genética , Ribonuclease III/genética , Alelos , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Estudos de Associação Genética , Genótipo , Humanos , MicroRNAs/genética , Risco
6.
Mol Cell ; 81(3): 546-557.e5, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33378643

RESUMO

Eukaryotic cells regulate 5'-triphosphorylated RNAs (ppp-RNAs) to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the γ phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR-1 (phosphatase that interacts with RNA and ribonucleoprotein particle 1) family of RNA polyphosphatases remove both the ß and γ phosphates from ppp-RNAs. Here, we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RNA-dependent RNA polymerases (RdRPs) and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and Dicer-independent Argonaute pathways and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Processamento Pós-Transcricional do RNA , RNA/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Monoéster Fosfórico Hidrolases/genética , Fosforilação , RNA/genética , Capuzes de RNA , /metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Espermatogênese , Especificidade por Substrato
7.
PLoS One ; 15(12): e0228233, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33347517

RESUMO

RNA interference (RNAi) plays key roles in post-transcriptional and chromatin modification levels as well as regulates various eukaryotic gene expressions which are involved in stress responses, development and maintenance of genome integrity during developmental stages. The whole mechanism of RNAi pathway is directly involved with the gene-silencing process by the interaction of Dicer-Like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) gene families and their regulatory elements. However, these RNAi gene families and their sub-cellular locations, functional pathways and regulatory components were not extensively investigated in the case of economically and nutritionally important fruit plant sweet orange (Citrus sinensis L.). Therefore, in silico characterization, gene diversity and regulatory factor analysis of RNA silencing genes in C. sinensis were conducted by using the integrated bioinformatics approaches. Genome-wide comparison analysis based on phylogenetic tree approach detected 4 CsDCL, 8 CsAGO and 4 CsRDR as RNAi candidate genes in C. sinensis corresponding to the RNAi genes of model plant Arabidopsis thaliana. The domain and motif composition and gene structure analyses for all three gene families exhibited almost homogeneity within the same group members. The Gene Ontology enrichment analysis clearly indicated that the predicted genes have direct involvement into the gene-silencing and other important pathways. The key regulatory transcription factors (TFs) MYB, Dof, ERF, NAC, MIKC_MADS, WRKY and bZIP were identified by their interaction network analysis with the predicted genes. The cis-acting regulatory elements associated with the predicted genes were detected as responsive to light, stress and hormone functions. Furthermore, the expressed sequence tag (EST) analysis showed that these RNAi candidate genes were highly expressed in fruit and leaves indicating their organ specific functions. Our genome-wide comparison and integrated bioinformatics analyses provided some necessary information about sweet orange RNA silencing components that would pave a ground for further investigation of functional mechanism of the predicted genes and their regulatory factors.


Assuntos
Citrus sinensis/genética , Regulação da Expressão Gênica de Plantas/genética , Interferência de RNA/fisiologia , Proteínas Argonauta/genética , Simulação por Computador , Etiquetas de Sequências Expressas , Frutas/metabolismo , Perfilação da Expressão Gênica/métodos , Genes de Plantas/genética , Genoma de Planta/genética , Família Multigênica/genética , Filogenia , Proteínas de Plantas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Ribonuclease III/genética , Fatores de Transcrição/metabolismo
8.
Nat Commun ; 11(1): 5445, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116115

RESUMO

Single-cell RNA sequencing studies on gene co-expression patterns could yield important regulatory and functional insights, but have so far been limited by the confounding effects of differentiation and cell cycle. We apply a tailored experimental design that eliminates these confounders, and report thousands of intrinsically covarying gene pairs in mouse embryonic stem cells. These covariations form a network with biological properties, outlining known and novel gene interactions. We provide the first evidence that miRNAs naturally induce transcriptome-wide covariations and compare the relative importance of nuclear organization, transcriptional and post-transcriptional regulation in defining covariations. We find that nuclear organization has the greatest impact, and that genes encoding for physically interacting proteins specifically tend to covary, suggesting importance for protein complex formation. Our results lend support to the concept of post-transcriptional RNA operons, but we further present evidence that nuclear proximity of genes may provide substantial functional regulation in mammalian single cells.


Assuntos
Núcleo Celular/genética , Redes Reguladoras de Genes , Mapas de Interação de Proteínas , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Variação Genética , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , RNA-Seq , Ribonuclease III/deficiência , Ribonuclease III/genética , Ribonuclease III/metabolismo , Análise de Célula Única , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma
9.
Proc Natl Acad Sci U S A ; 117(38): 23932-23941, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900951

RESUMO

DICER is a key enzyme in microRNA (miRNA) biogenesis. Here we show that aerobic exercise training up-regulates DICER in adipose tissue of mice and humans. This can be mimicked by infusion of serum from exercised mice into sedentary mice and depends on AMPK-mediated signaling in both muscle and adipocytes. Adipocyte DICER is required for whole-body metabolic adaptations to aerobic exercise training, in part, by allowing controlled substrate utilization in adipose tissue, which, in turn, supports skeletal muscle function. Exercise training increases overall miRNA expression in adipose tissue, and up-regulation of miR-203-3p limits glycolysis in adipose under conditions of metabolic stress. We propose that exercise training-induced DICER-miR-203-3p up-regulation in adipocytes is a key adaptive response that coordinates signals from working muscle to promote whole-body metabolic adaptations.


Assuntos
Tecido Adiposo/metabolismo , RNA Helicases DEAD-box/metabolismo , Exercício Físico/fisiologia , Ribonuclease III/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Adaptação Fisiológica/fisiologia , Adipócitos/metabolismo , Animais , Células Cultivadas , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Feminino , Glicólise , Humanos , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Condicionamento Físico Animal , Ribonuclease III/deficiência , Ribonuclease III/genética
10.
Mol Cell Biol ; 40(20)2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32778571

RESUMO

Many proteins, including DICER1 and hAgo2, are involved in the biogenesis of microRNAs (miRNAs). Whether hAgo2 regulates DICER1 expression is unknown. Exogenously overexpressed hAgo2 suppressed DICER1 expression at the levels of both protein and mRNA, and the reduction in hAgo2 expression enhanced DICER1 expression. Precursor miRNA processing mediated by DICER1 was also modulated by hAgo2. However, hAgo2 protein did not suppress DICER1 promoter activity. Therefore, hAgo2 protein probably regulates DICER1 expression at the posttranscriptional level. Indeed, hAgo2 protein inhibited the reporter assay of the DICER1 mRNA 3' untranslated region (3'-UTR). Previous reports have demonstrated that miRNAs (e.g., let-7 and miR-103/107) inhibited DICER1 expression posttranscriptionally. However, hAgo2 still suppressed DICER1 expression in the cells depleted of these miRNAs. Moreover, the reporter activities of the DICER1 mRNA 3'-UTR without these miRNA binding sites were still suppressed by hAgo2. Therefore, in addition to an miRNA-dependent pathway, hAgo2 can also modulate DICER1 expression through an miRNA-independent mechanism. Downregulation of DICER1 expression was further proven to be dependent on both hAgo2 and AUF1 proteins. Interactions of hAgo2 and AUF1 proteins were demonstrated by the coimmunoprecipitation assay. As expected, hAgo2 could not suppress the DICER1 mRNA 3'-UTR reporter with a mutation in the potential AUF1-binding site. Thus, downregulation of DICER1 expression through the 3'-UTR requires both hAgo2 and AUF1.


Assuntos
Proteínas Argonauta/genética , RNA Helicases DEAD-box/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Neoplasias/genética , Ribonuclease III/genética , Regiões 3' não Traduzidas/genética , Células A549 , Proteínas Argonauta/metabolismo , Sítios de Ligação/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0/genética , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Humanos , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ribonuclease III/metabolismo
11.
PLoS Genet ; 16(7): e1008611, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658892

RESUMO

Epimutations in fungal pathogens are emerging as novel phenomena that could explain the fast-developing resistance to antifungal drugs and other stresses. These epimutations are generated by RNA interference (RNAi) mechanisms that transiently silence specific genes to overcome stressful stimuli. The early-diverging fungus Mucor circinelloides exercises a fine control over two interacting RNAi pathways to produce epimutants: the canonical RNAi pathway and a new RNAi degradative pathway. The latter is considered a non-canonical RNAi pathway (NCRIP) because it relies on RNA-dependent RNA polymerases (RdRPs) and a novel ribonuclease III-like named R3B2 to degrade target transcripts. Here in this work, we uncovered the role of NCRIP in regulating virulence processes and transposon movements through key components of the pathway, RdRP1 and R3B2. Mutants in these genes are unable to launch a proper virulence response to macrophage phagocytosis, resulting in a decreased virulence potential. The transcriptomic profile of rdrp1Δ and r3b2Δ mutants revealed a pre-exposure adaptation to the stressful phagosomal environment even when the strains are not confronted by macrophages. These results suggest that NCRIP represses key targets during regular growth and releases its control when a stressful environment challenges the fungus. NCRIP interacts with the RNAi canonical core to protect genome stability by controlling the expression of centromeric retrotransposable elements. In the absence of NCRIP, these retrotransposons are robustly repressed by the canonical RNAi machinery; thus, supporting the antagonistic role of NCRIP in containing the epimutational pathway. Both interacting RNAi pathways might be essential to govern host-pathogen interactions through transient adaptations, contributing to the unique traits of the emerging infection mucormycosis.


Assuntos
Mucorales/genética , Mucormicose/genética , Interferência de RNA , Ribonuclease III/genética , Antifúngicos/farmacologia , Farmacorresistência Fúngica/genética , Epigênese Genética/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Instabilidade Genômica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Mucorales/patogenicidade , Mucormicose/microbiologia , Mutação/genética , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Virulência/genética
12.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641477

RESUMO

Positive-strand RNA [(+)RNA] viruses are important pathogens of humans, animals, and plants and replicate inside host cells by coopting numerous host factors and subcellular membranes. To gain insights into the assembly of viral replicase complexes (VRCs) and dissect the roles of various lipids and coopted host factors, we have reconstituted Tomato bushy stunt virus (TBSV) replicase using artificial giant unilamellar vesicles (GUVs). We demonstrate that reconstitution of VRCs on GUVs with endoplasmic reticulum (ER)-like phospholipid composition results in a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)RNA viruses. TBSV VRCs assembled on GUVs provide significant protection of the double-stranded RNA (dsRNA) replication intermediate against the dsRNA-specific RNase III. The lipid compositions of GUVs have pronounced effects on in vitro TBSV replication, including (-) and (+)RNA synthesis. The GUV-based assay has led to the discovery of the critical role of phosphatidylserine in TBSV replication and a novel role for phosphatidylethanolamine in asymmetrical (+)RNA synthesis. The GUV-based assay also showed stimulatory effects by phosphatidylinositol-3-phosphate [PI(3)P] and ergosterol on TBSV replication. We demonstrate that eEF1A and Hsp70 coopted replicase assembly factors, Vps34 phosphatidylinositol 3-kinase (PI3K) and the membrane-bending ESCRT factors, are required for reconstitution of the active TBSV VRCs in GUVs, further supporting that the novel GUV-based in vitro approach recapitulates critical steps and involves essential coopted cellular factors of the TBSV replication process. Taken together, this novel GUV assay will be highly suitable to dissect the functions of viral and cellular factors in TBSV replication.IMPORTANCE Understanding the mechanism of replication of positive-strand RNA viruses, which are major pathogens of plants, animals, and humans, can lead to new targets for antiviral interventions. These viruses subvert intracellular membranes for virus replication and coopt numerous host proteins, whose functions during virus replication are not yet completely defined. To dissect the roles of various host factors in Tomato bushy stunt virus (TBSV) replication, we have developed an artificial giant unilamellar vesicle (GUV)-based replication assay. The GUV-based in vitro approach recapitulates critical steps of the TBSV replication process. GUV-based reconstitution of the TBSV replicase revealed the need for a complex mixture of phospholipids, especially phosphatidylserine and phosphatidylethanolamine, in TBSV replication. The GUV-based approach will be useful to dissect the functions of essential coopted cellular factors.


Assuntos
RNA de Cadeia Dupla/genética , Tombusvirus/genética , Lipossomas Unilamelares/metabolismo , Proteínas Virais/genética , Bioensaio , Linhagem Celular , Retículo Endoplasmático/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ergosterol/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Células Vegetais/metabolismo , Células Vegetais/virologia , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/metabolismo , Lipossomas Unilamelares/química , Proteínas Virais/metabolismo , Replicação Viral
13.
Medicine (Baltimore) ; 99(27): e20806, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32629665

RESUMO

RATIONALE: DICER1 syndrome is an autosomal-dominant tumor predisposition syndrome associated with numerous cancerous and noncancerous conditions. The most common sex cord-stromal tumor associated with DICER1 syndrome is Sertoli-Leydig cell tumor of the ovary (SLCT), which is extremely unusual and accounts for < 0.5% of all ovarian neoplasms. SLCT predominantly affects adolescents and young female adults. To date, there are only a few case reports of ovarian SLCT with underlying germline DICER1 mutations. The diagnosis and treatment of this rare malignancy remains challenging in the clinic mainly due to its rarity and varied presentation. PATIENT CONCERNS: A 21-year-old Chinese girl (proband) was admitted in hospital for experiencing a lower abdominal pain and irregular vaginal bleeding for half a year. She was initially diagnosed with abdominal cavity mass prior to surgical operation. The other 20-year-old patient is the younger sister of the proband, who was diagnosed with ovarian cysts and had irregular menstruation and amenorrhea for 4 months. The elder sister underwent an uncomplicated bilateral ovarian tumor resection. Given a high degree of malignancy, comprehensive staged fertility-preserving surgery, including left adnexectomy, omentectomy, pelvic, and para-aortic lymphadenectomy, was performed. Since the other patient requested to maintain her fertility, tumor resection was only conducted in the right ovary. DIAGNOSES: The elder sister was diagnosed as poorly differentiated SLCT accompanied with heterologous stage IC rhabdomyosarcoma (RMS) based on its typical pathology features and molecular characteristics from immunohistochemistry (IHC) staining. The younger sister was diagnosed as poorly differentiated SLCT. Targeted next-generation sequencing (NGS) detected DICER1 mutation in the plasma samples and postoperative tumor tissues of both patients. INTERVENTIONS: Both patients underwent surgical tumor resection, followed by combination chemotherapy with bleomycin, etoposide, and cisplatin for 4 cycles. OUTCOMES: Patients received the above clinical interventions but eventually died from disease recurrence. The elder sister died from disease relapse after one and a half years postsurgery. The younger sister had a relapse of the disease 1 year later, but she refused the comprehensive staged surgery and died from disease relapse quickly. LESSONS: Ovarian SLCT patients with DICER1 mutations and a family history have a high degree of malignancy and are associated with a poor prognosis. With ongoing research efforts on DICER1 mutations, genetic screening and counselling on a regular basis is recommended for predicting potential future cancer risk of individuals with DICER1 syndrome family history.


Assuntos
RNA Helicases DEAD-box/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ribonuclease III/genética , Tumor de Células de Sertoli-Leydig/genética , Tumor de Células de Sertoli-Leydig/patologia , Feminino , Humanos , Neoplasias Ovarianas/terapia , Tumor de Células de Sertoli-Leydig/terapia , Irmãos , Adulto Jovem
14.
PLoS Genet ; 16(6): e1008808, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32497036

RESUMO

Metastasis is responsible for 90% of human cancer mortality, yet it remains a challenge to model human cancer metastasis in vivo. Here we describe mouse models of high-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), the most common and deadliest human ovarian cancer type. Mice genetically engineered to harbor Dicer1 and Pten inactivation and mutant p53 robustly replicate the peritoneal metastases of human HGSC with complete penetrance. Arising from the fallopian tube, tumors spread to the ovary and metastasize throughout the pelvic and peritoneal cavities, invariably inducing hemorrhagic ascites. Widespread and abundant peritoneal metastases ultimately cause mouse deaths (100%). Besides the phenotypic and histopathological similarities, mouse HGSCs also display marked chromosomal instability, impaired DNA repair, and chemosensitivity. Faithfully recapitulating the clinical metastases as well as molecular and genomic features of human HGSC, this murine model will be valuable for elucidating the mechanisms underlying the development and progression of metastatic ovarian cancer and also for evaluating potential therapies.


Assuntos
Antineoplásicos/farmacologia , Cistadenocarcinoma Seroso/genética , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/genética , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Instabilidade Cromossômica , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/secundário , RNA Helicases DEAD-box/genética , Reparo do DNA , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Estudos de Viabilidade , Feminino , Humanos , Camundongos , Camundongos Knockout , Mutação , Gradação de Tumores , Metástase Neoplásica/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/secundário , Cultura Primária de Células , Ribonuclease III/genética , Proteína Supressora de Tumor p53/genética
15.
Nucleic Acids Res ; 48(12): 6839-6854, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32449937

RESUMO

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.


Assuntos
Proteínas de Arabidopsis/genética , MicroRNAs/genética , RNA Helicases/genética , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genética , Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/genética , Exossomos/genética , Regulação da Expressão Gênica de Plantas/genética , Mutação/genética , Precursores de RNA/genética , Estabilidade de RNA/genética , Ribonuclease III/genética
16.
Zhonghua Bing Li Xue Za Zhi ; 49(5): 441-447, 2020 May 08.
Artigo em Chinês | MEDLINE | ID: mdl-32392927

RESUMO

Objective: To investigate DICER1 hotspot mutations in ovarian Sertoli-Leydig cell tumor (SLCT) and its associated clinicopathological features. Methods: Forty-three SLCTs and 40 other sex cord-stromal tumors (SCSTs) diagnosed between 2010 and 2017 at Fudan University Shanghai Cancer Center were examined for somatic DICER1 hotspot mutations by Sanger sequencing. The associations between mutation status and clinicopathological features, including patient age, tumor differentiation and recurrence, were analyzed. Results: Somatic DICER1 mutations were found in 51% (22/43) of SLCTs, while none in the other 40 SCSTs. The most common mutation of DICER1 was p.D1709N in exon 24 (41%, 9/22) and the second most common mutation of DICER1 was p.E1813K in exon 25 (14%, 3/22). A novel frameshift mutation (c.5464delG, p.M1837fs*16) was identified in one SLCT with microcystic pattern. Mutations were more likely to occur in patients under forty years of age (P=0.046), whereas no significant associations were found between DICER1 mutations and clinical symptoms, morphology or tumor recurrence. Conclusions: Somatic DCIER1 hotspot mutations are specifically found in SLCT and may serve as an ancillary marker in differential diagnosis of SLCT from other SCST. The mutations occur more often in young patients (<40 years old). Additional studies are warranted to examine the associations between DICER1 mutations and clinicopathological features and prognosis of SLCT.


Assuntos
RNA Helicases DEAD-box/genética , Neoplasias Ovarianas , Ribonuclease III/genética , Tumor de Células de Sertoli-Leydig , Adulto , China , Feminino , Humanos , Mutação , Neoplasias Ovarianas/genética , Tumor de Células de Sertoli-Leydig/genética , Tumores do Estroma Gonadal e dos Cordões Sexuais
17.
Mol Cell ; 78(2): 303-316.e4, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32302542

RESUMO

Nuclear processing of most miRNAs is mediated by Microprocessor, comprised of RNase III enzyme Drosha and its cofactor DGCR8. Here, we uncover a hidden layer of Microprocessor regulation via studies of Dicer-independent mir-451, which is clustered with canonical mir-144. Although mir-451 is fully dependent on Drosha/DGCR8, its short stem and small terminal loop render it an intrinsically weak Microprocessor substrate. Thus, it must reside within a cluster for normal biogenesis, although the identity and orientation of its neighbor are flexible. We use DGCR8 tethering assays and operon structure-function assays to demonstrate that local recruitment and transfer of Microprocessor enhances suboptimal substrate processing. This principle applies more broadly since genomic analysis indicates suboptimal canonical miRNAs are enriched in operons, and we validate several of these experimentally. Proximity-based enhancement of suboptimal hairpin processing provides a rationale for genomic retention of certain miRNA operons and may explain preferential evolutionary emergence of miRNA operons.


Assuntos
Genômica , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Núcleo Celular/genética , Humanos , Processamento Pós-Transcricional do RNA/genética
18.
Virchows Arch ; 477(3): 375-382, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32193603

RESUMO

Congenital pulmonary airway malformation (CPAM) occurs most commonly in infants. It is divided into 5 types. The most common types 1 and 2 are cystic, type 0 presents as bronchial buds without alveolar tissue, most likely corresponding to alveolar dysgenesis, while type 3 is composed of branching bronchioles and appears as a solid lesion. A defect in the epithelial-mesenchymal crosstalk might be the underlying mechanism for all. Type 4 is a peripheral cystic lesion with a thin cyst wall covered by pneumocytes. CPAM 4 has been mixed up with pleuropulmonary blastoma (PPB) type I and some authors question its existence. We investigated five cases of CPAM type 4 for the presence or absence of rhabdomyoblasts, and for markers associated with CPAM development. In addition, all cases were evaluated for mutations within the Dicer gene and for mutations of the RAS family of oncogenes. All five cases showed smooth muscle actin and desmin-positive cells; however, only one case showed a few cells positive for MyoD. The same case showed a mutation of Dicer 1. All cases were negative for mutations of the RAS family of genes. Fibroblast growth factor 10 was similarly expressed in all cases, and thus cannot be used to differentiate CPAM4 from PPB-I. Low expression of the proliferation marker Ki67 was seen in our CPAM 4 cases and the probable PPB-I case. YingYang-1 protein seems to play an active role in the development of PPB-I. CPAM 4 can be separated from PPB-I based on the presence of rhabdomyoblasts and mutations in Dicer 1 gene. These cells might not be numerous; therefore, all available tissue has to be evaluated. As CPAM 4 morphologically looks very similar to PPB-I, it might be speculated, that there exists a potential for progression from CPAM 4 to PPB-I, by acquiring somatic mutations in Dicer 1.


Assuntos
Malformação Adenomatoide Cística Congênita do Pulmão/patologia , RNA Helicases DEAD-box/genética , Blastoma Pulmonar/etiologia , Blastoma Pulmonar/genética , Ribonuclease III/genética , Adolescente , Biomarcadores Tumorais/genética , Malformação Adenomatoide Cística Congênita do Pulmão/complicações , Malformação Adenomatoide Cística Congênita do Pulmão/diagnóstico , Diagnóstico Diferencial , Progressão da Doença , Feminino , Genes ras , Humanos , Lactente , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Adulto Jovem
19.
Mol Cell ; 78(2): 317-328.e6, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32191872

RESUMO

MicroRNAs (miRNAs) are sequentially processed by two RNase III enzymes, Drosha and Dicer. miR-451 is the only known miRNA whose processing bypasses Dicer and instead relies on the slicer activity of Argonaute-2 (Ago2). miR-451 is highly conserved in vertebrates and regulates erythrocyte maturation, where it becomes the most abundant miRNA. However, the basis for the non-canonical biogenesis of miR-451 is unclear. Here, we show that Ago2 is less efficient than Dicer in processing pre-miRNAs, but this deficit is overcome when miR-144 represses Dicer in a negative-feedback loop during erythropoiesis. Loss of miR-144-mediated Dicer repression in zebrafish embryos and human cells leads to increased canonical miRNA production and impaired miR-451 maturation. Overexpression of Ago2 rescues some of the defects of miR-451 processing. Thus, the evolution of Ago2-dependent processing allows miR-451 to circumvent the global repression of canonical miRNAs elicited, in part, by the miR-144 targeting of Dicer during erythropoiesis.


Assuntos
Proteínas Argonauta/genética , Eritropoese/genética , MicroRNAs/genética , Animais , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Interferência de RNA , Ribonuclease III/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
20.
Mol Cell ; 78(3): 423-433.e5, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32220645

RESUMO

A commencing and critical step in miRNA biogenesis involves processing of pri-miRNAs in the nucleus by Microprocessor. An important, but not completely understood, question is how Drosha, the catalytic subunit of Microprocessor, binds pri-miRNAs and correctly specifies cleavage sites. Here we report the cryoelectron microscopy structures of the Drosha-DGCR8 complex with and without a pri-miRNA. The RNA-bound structure provides direct visualization of the tertiary structure of pri-miRNA and shows that a helix hairpin in the extended PAZ domain and the mobile basic (MB) helix in the RNase IIIa domain of Drosha coordinate to recognize the single-stranded to double-stranded junction of RNA, whereas the dsRNA binding domain makes extensive contacts with the RNA stem. Furthermore, the RNA-free structure reveals an autoinhibitory conformation of the PAZ helix hairpin. These findings provide mechanistic insights into pri-miRNA cleavage site selection and conformational dynamics governing pri-miRNA recognition by the catalytic component of Microprocessor.


Assuntos
MicroRNAs/química , MicroRNAs/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Animais , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Spodoptera/citologia
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