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Drosophila is a well-established insect model system for studying various physiological phenomena and developmental processes, with a focus on gene regulation. Drosophila development is controlled by programmed regulatory mechanisms specific to individual tissues. When key developmental processes are shared among various insects, the associated regulatory networks are believed to be conserved across insects. Thus, studies of developmental regulation in Drosophila have substantially contributed to our understanding of insect development. Over the past two decades, studies on microRNAs (miRNAs) in Drosophila have revealed their crucial regulatory roles in various developmental processes. This review focuses on the biological roles of miRNAs in specific tissues and processes associated with Drosophila development. Additionally, as a future direction, we discuss sequencing technologies that can analyze the interactions between miRNAs and their target genes, with the aim of enhancing miRNA studies in Drosophila development.
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ABSTRACT: The transcription factor RUNX1 is a master regulator of hematopoiesis and is frequently mutated in myeloid malignancies. Mutations in its runt homology domain (RHD) frequently disrupt DNA binding and result in loss of RUNX1 function. However, it is not clearly understood how other RUNX1 mutations contribute to disease development. Here, we characterized RUNX1 mutations outside of the RHD. Our analysis of the patient data sets revealed that mutations within the C-terminus frequently occur in hematopoietic disorders. Remarkably, most of these mutations were nonsense or frameshift mutations and were predicted to be exempt from nonsense-mediated messenger RNA decay. Therefore, this class of mutation is projected to produce DNA-binding proteins that contribute to the pathogenesis in a distinct manner. To model this, we introduced the RUNX1R320∗ mutation into the endogenous gene locus and demonstrated the production of RUNX1R320∗ protein. Expression of RUNX1R320∗ resulted in the disruption of RUNX1 regulated processes such as megakaryocytic differentiation, through a transcriptional signature different from RUNX1 depletion. To understand the underlying mechanisms, we used Global RNA Interactions with DNA by deep sequencing (GRID-seq) to examine enhancer-promoter connections. We identified widespread alterations in the enhancer-promoter networks within RUNX1 mutant cells. Additionally, we uncovered enrichment of RUNX1R320∗ and FOXK2 binding at the MYC super enhancer locus, significantly upregulating MYC transcription and signaling pathways. Together, our study demonstrated that most RUNX1 mutations outside the DNA-binding domain are not subject to nonsense-mediated decay, producing protein products that act in concert with additional cofactors to dysregulate hematopoiesis through mechanisms distinct from those induced by RUNX1 depletion.
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Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core , Mutação , Regiões Promotoras Genéticas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Humanos , Diferenciação Celular/genética , Elementos Facilitadores Genéticos , Células Sanguíneas/metabolismo , Redes Reguladoras de Genes , Regulação da Expressão GênicaRESUMO
The accumulation of misfolded and aggregated proteins is a hallmark of neurodegenerative proteinopathies. Although multiple genetic loci have been associated with specific neurodegenerative diseases (NDs), molecular mechanisms that may have a broader relevance for most or all proteinopathies remain poorly resolved. In this study, we developed a multi-layered network expansion (MLnet) model to predict protein modifiers that are common to a group of diseases and, therefore, may have broader pathophysiological relevance for that group. When applied to the four NDs Alzheimer's disease (AD), Huntington's disease, and spinocerebellar ataxia types 1 and 3, we predicted multiple members of the insulin pathway, including PDK1, Akt1, InR, and sgg (GSK-3ß), as common modifiers. We validated these modifiers with the help of four Drosophila ND models. Further evaluation of Akt1 in human cell-based ND models revealed that activation of Akt1 signaling by the small molecule SC79 increased cell viability in all models. Moreover, treatment of AD model mice with SC79 enhanced their long-term memory and ameliorated dysregulated anxiety levels, which are commonly affected in AD patients. These findings validate MLnet as a valuable tool to uncover molecular pathways and proteins involved in the pathophysiology of entire disease groups and identify potential therapeutic targets that have relevance across disease boundaries. MLnet can be used for any group of diseases and is available as a web tool at http://ssbio.cau.ac.kr/software/mlnet.
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Doença de Alzheimer , Doença de Huntington , Deficiências na Proteostase , Animais , Humanos , Camundongos , Doença de Alzheimer/genética , Glicogênio Sintase Quinase 3 beta , Doença de Huntington/genética , Transdução de SinaisRESUMO
Animal growth is controlled by a variety of external and internal factors during development. The steroid hormone ecdysone plays a critical role in insect development by regulating the expression of various genes. In this study, we found that fat body-specific expression of miR-276a, an ecdysone-responsive microRNA (miRNA), led to a decrease in the total mass of the larval fat body, resulting in significant growth reduction in Drosophila. Changes in miR-276a expression also affected the proliferation of Drosophila S2 cells. Furthermore, we found that the insulin-like receptor (InR) is a biologically relevant target gene regulated by miR-276a-3p. In addition, we found that miR-276a-3p is upregulated by the canonical ecdysone signalling pathway involving the ecdysone receptor and broad complex. A reduction in cell proliferation caused by ecdysone was compromised by blocking miR-276a-3p activity. Thus, our results suggest that miR-276a-3p is involved in ecdysone-mediated growth reduction by controlling InR expression in the insulin signalling pathway.
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Proteínas de Drosophila , Insulinas , MicroRNAs , Animais , Drosophila/genética , Ecdisona/metabolismo , MicroRNAs/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Drosophila/genética , Insulinas/genética , Insulinas/metabolismo , Drosophila melanogaster/genéticaRESUMO
The hematopoietic system plays a crucial role in immune defense response and normal development, and it is regulated by various factors from other tissues. The dysregulation of hematopoiesis is associated with melanotic mass formation; however, the molecular mechanisms underlying this process are poorly understood. Here, we observed that the overexpression of miR-274 in the fat body resulted in the formation of melanotic masses. Moreover, abnormal activation of the JNK and JAK/STAT signaling pathways was linked to these consequences. In addition to this defect, miR-274 overexpression in the larval fat body decreased the total tissue size, leading to a reduction in body weight. miR-274-5p was found to directly suppress the expression of found-in-neurons (fne), which encodes an RNA-binding protein. Similar to the effects of miR-274 overexpression, fne depletion led to melanotic mass formation and growth reduction. Collectively, miR-274 plays a regulatory role in the fne-JNK signaling axis in melanotic mass formation and growth control.
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Basement membranes (BMs) play important roles under various physiological conditions in animals, including ecdysozoans. During development, BMs undergo alterations through diverse intrinsic and extrinsic regulatory mechanisms; however, the full complement of pathways controlling these changes remain unclear. Here, we found that fat body-overexpression of Drosophila miR-263b, which is highly expressed during the larval-to-pupal transition, resulted in a decrease in the overall size of the larval fat body, and ultimately, in a severe growth defect accompanied by a reduction in cell proliferation and cell size. Interestingly, we further observed that a large proportion of the larval fat body cells were prematurely disassociated from each other. Moreover, we present evidence that miR-263b-5p suppresses the main component of BMs, Laminin A (LanA). Through experiments using RNA interference (RNAi) of LanA, we found that its depletion phenocopied the effects in miR-263b-overexpressing flies. Overall, our findings suggest a potential role for miR-263b in developmental growth and cell association by suppressing LanA expression in the Drosophila fat body.
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The Notch signaling pathway plays a central role in the development of various organisms. However, dysregulation of microRNAs (miRNAs), which are crucial regulators of gene expression, can disrupt signaling pathways at all stages of development. Although Notch signaling is involved in wing development in Drosophila, the mechanism underlying miRNA-based regulation of the Notch signaling pathway is unclear. Here, we report that loss of Drosophila miR-252 increases the size of adult wings, whereas the overexpression of miR-252 in specific compartments of larval wing discs leads to patterning defects in the adult wings. The miR-252 overexpression-induced wing phenotypes were caused by aberrant Notch signaling with intracellular accumulation of the full-length Notch receptor during development, which could be due to defects in intracellular Notch trafficking associated with its recycling to the plasma membrane and autophagy-mediated degradation. Moreover, we identified Rab6 as a direct target of miR-252-5p; Rab6 encodes a small Ras-like GTPase that regulates endosomal trafficking pathways. Consistent with this finding, RNAi-mediated downregulation of Rab6 led to similar defects in both wing patterning and Notch signaling. Notably, co-overexpression of Rab6 completely rescued the wing phenotype associated with miR-252 overexpression, further supporting that Rab6 is a biologically relevant target of miR-252-5p in the context of wing development. Thus, our data indicate that the miR-252-5p-Rab6 regulatory axis is involved in Drosophila wing development by controlling the Notch signaling pathway.
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It is increasingly evident that various RNAs can bind chromatin to regulate gene expression and genome organization. Here we adapted a sequencing-based technique to profile RNA-chromatin interactions at a genome-wide scale in Arabidopsis seedlings. We identified more than 10,000 RNA-chromatin interactions mediated by protein-coding RNAs and non-coding RNAs. Cis and intra-chromosomal interactions are mainly mediated by protein-coding RNAs, whereas inter-chromosomal interactions are primarily mediated by non-coding RNAs. Many RNA-chromatin interactions tend to positively correlate with DNA-DNA interactions, suggesting their mutual influence and reinforcement. We further show that some RNA-chromatin interactions undergo alterations in response to biotic and abiotic stresses and that altered RNA-chromatin interactions form co-regulatory networks. Our study provides a global view on RNA-chromatin interactions in Arabidopsis and a rich resource for future investigations of regulatory roles of RNAs in gene expression and genome organization.
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Arabidopsis/genética , Arabidopsis/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , RNA de Plantas/genética , RNA de Plantas/metabolismo , Plântula/genética , Plântula/metabolismo , Estresse Fisiológico/genéticaRESUMO
Retrotransposons are populated in vertebrate genomes, and when active, are thought to cause genome instability with potential benefit to genome evolution. Retrotransposon-derived RNAs are also known to give rise to small endo-siRNAs to help maintain heterochromatin at their sites of transcription; however, as not all heterochromatic regions are equally active in transcription, it remains unclear how heterochromatin is maintained across the genome. Here, we address these problems by defining the origins of repeat-derived RNAs and their specific chromatin locations in Drosophila S2 cells. We demonstrate that repeat RNAs are predominantly derived from active gypsy elements and processed by Dcr-2 into small RNAs to help maintain pericentromeric heterochromatin. We also show in cultured S2 cells that synthetic repeat-derived endo-siRNA mimics are sufficient to rescue Dcr-2-deficiency-induced defects in heterochromatin formation in interphase and chromosome segregation during mitosis, demonstrating that active retrotransposons are required for stable genetic inheritance.
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Divisão Celular/genética , Heterocromatina , Retroelementos , Animais , Centrômero , Segregação de Cromossomos , Drosophila/genética , Proteínas de Drosophila/genética , Eucromatina , Sequenciamento de Nucleotídeos em Larga Escala , RNA Helicases/genética , RNA Interferente Pequeno , Ribonuclease III/genéticaRESUMO
Cap1 2'-O-ribose methyltransferase (CMTR1) modifies RNA transcripts containing the 7-methylguanosine cap via 2'-O-ribose methylation of the first transcribed nucleotide, yielding cap1 structures. However, the role of CMTR1 in small RNA-mediated gene silencing remains unknown. Here, we identified and characterized a Drosophila CMTR1 gene (dCMTR1) mutation. We found that the catalytic activity of dCMTR1 was involved in the biogenesis of small interfering RNAs (siRNAs) but not microRNAs. Additionally, dCMTR1 interacted with R2D2, a key component for the assembly of the RNA-induced silencing complex (RISC) containing Argonaute 2 (Ago2). Consistent with this finding, loss of dCMTR1 function impaired RISC assembly by inhibiting the unwinding of Ago2-bound siRNA duplexes, thus preventing the retention of the guide strand. Moreover, dCMTR1 is unlikely to modify siRNAs during RISC assembly. Collectively, our data indicate that dCMTR1 is a positive regulator of the small RNA pathway associated with Ago2 with roles in both siRNA biogenesis and RISC assembly.
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Drosophila/metabolismo , Metiltransferases , RNA Interferente Pequeno , Complexo de Inativação Induzido por RNA , Animais , Proteínas Argonautas/metabolismo , Drosophila/genética , Proteínas de Drosophila/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , MicroRNAs/metabolismo , Mutação , Interferência de RNA , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/química , Complexo de Inativação Induzido por RNA/biossíntese , Complexo de Inativação Induzido por RNA/química , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
The steroid hormone ecdysone is the central regulator of insect metamorphosis, during which a growing, immature larva is remodeled, through pupal stages, to a reproductive adult. However, the underlying mechanisms of ecdysone-mediated metamorphosis remain to be fully elucidated. Here, we identified metamorphosis-associated microRNAs (miRNAs) and their potential targets by cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 protein in Drosophila. Interestingly, miR-8-3p targeted five Vha genes encoding distinct subunits of vacuolar H+ -ATPase (V-ATPase), which has a vital role in the organellar acidification. The expression of ecdysone-responsive miR-8-3p is normally downregulated during Drosophila metamorphosis, but temporary overexpression of miR-8-3p in the whole body at the end of larval development led to defects in metamorphosis and survival, hallmarks of aberrant ecdysone signaling. In addition, miR-8-3p was expressed in the prothoracic gland (PG), which produces and releases ecdysone in response to prothoracicotropic hormone (PTTH). Notably, overexpression of miR-8-3p or knockdown of its Vha targets in the PG resulted in larger than normal, ecdysone-deficient larvae that failed to develop into the pupal stage but could be rescued by ecdysone feeding. Moreover, these animals showed defective PTTH signaling with a concomitant decrease in the expression of ecdysone biosynthetic genes. We also demonstrated that the regulatory network between the conserved miR-8-3p/miR-200 family and V-ATPase was functional in human cells. Consequently, our data indicate that the coordinated regulation of V-ATPase subunits by miR-8-3p is involved in Drosophila metamorphosis by controlling the ecdysone biosynthesis.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Ecdisona/biossíntese , Metamorfose Biológica , MicroRNAs/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de Drosophila/genética , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.
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Cromatina/metabolismo , Protaminas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/fisiologia , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Fertilização/genética , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/metabolismo , Oócitos/fisiologia , Fosforilação , Protamina Quinase/genética , Protamina Quinase/metabolismo , Protaminas/genética , Proteínas Serina-Treonina Quinases/fisiologia , Splicing de RNA/genética , Splicing de RNA/fisiologia , Espermatozoides/metabolismo , Fatores de Transcrição/metabolismo , Zigoto/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Chromatin in higher eukaryotic nuclei is extensively bound by various RNA species. We recently developed a method for in situ capture of global RNA interactions with DNA by deep sequencing (GRID-seq) of fixed permeabilized nuclei that allows identification of the entire repertoire of chromatin-associated RNAs in an unbiased manner. The experimental design of GRID-seq is related to those of two recently published strategies (MARGI (mapping RNA-genome interactions) and ChAR-seq (chromatin-associated RNA sequencing)), which also use a bivalent linker to ligate RNA and DNA in proximity. Importantly, however, GRID-seq also implements a combined experimental and computational approach to control nonspecific RNA-DNA interactions that are likely to occur during library construction, which is critical for accurate interpretation of detected RNA-DNA interactions. GRID-seq typically finds both coding and non-coding RNAs (ncRNAs) that interact with tissue-specific promoters and enhancers, especially super-enhancers, from which a global promoter-enhancer connectivity map can be deduced. Here, we provide a detailed protocol for GRID-seq that includes nuclei preparation, chromatin fragmentation, RNA and DNA in situ ligation with a bivalent linker, PCR amplification and high-throughput sequencing. To further enhance the utility of GRID-seq, we include a pipeline for data analysis, called GridTools, into which key steps such as background correction and inference of genomic element proximity are integrated. For researchers experienced in molecular biology with minimal bioinformatics skills, the protocol typically takes 4-5 d from cell fixation to library construction and 2-3 d for data processing.
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Cromatina/genética , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Animais , Núcleo Celular/genética , Cromatina/química , Cromatina/metabolismo , Drosophila/genética , Biblioteca Gênica , Camundongos , Camundongos Endogâmicos , Células-Tronco Embrionárias Murinas/fisiologia , Regiões Promotoras Genéticas , RNA/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Fluxo de TrabalhoRESUMO
Eukaryotic genomes are known to prevalently transcribe diverse classes of RNAs, virtually all of which, including nascent RNAs from protein-coding genes, are now recognized to have regulatory functions in gene expression, suggesting that RNAs are both the products and the regulators of gene expression. Their functions must enlist specific RNA-binding proteins (RBPs) to execute their regulatory activities, and recent evidence suggests that nearly all biochemically defined chromatin regions in the human genome, whether defined for gene activation or silencing, have the involvement of specific RBPs. Interestingly, the boundary between RNA- and DNA-binding proteins is also melting, as many DNA-binding proteins traditionally studied in the context of transcription are able to bind RNAs, some of which may simultaneously bind both DNA and RNA to facilitate network interactions in three-dimensional (3D) genome. In this review, we focus on RBPs that function at chromatin levels, with particular emphasis on their mechanisms of action in regulated gene expression, which is intended to facilitate future functional and mechanistic dissection of chromatin-associated RBPs.
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The steroid hormone ecdysone has a central role in the developmental transitions of insects through its control of responsive protein-coding and microRNA (miRNA) gene expression. However, the complete regulatory network controlling the expression of these genes remains to be elucidated. In this study, we performed cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 (Ago1) protein, the core effector of the miRNA pathway, in Drosophila S2 cells. We found that regulatory interactions between miRNAs and their cognate targets were substantially altered by Ago1 in response to ecdysone signaling. Additionally, during the larva-to-adult metamorphosis, miR-252-5p was up-regulated via the canonical ecdysone-signaling pathway. Moreover, we provide evidence that miR-252-5p targets Abelson interacting protein ( Abi) to decrease the protein levels of cyclins A and B, controlling the cell cycle. Overall, our data suggest a potential role for the ecdysone/miR-252-5p/Abi regulatory axis partly in cell-cycle control during metamorphosis in Drosophila.-Lim, D.-H., Lee, S., Han, J. Y., Choi, M.-S., Hong, J.-S., Seong, Y., Kwon, Y.-S., Lee, Y. S. Ecdysone-responsive microR-252-5p controls the cell cycle by targeting Abi in Drosophila.
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Proteínas de Transporte/metabolismo , Ciclo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Ecdisona/metabolismo , MicroRNAs/metabolismo , Animais , Proteínas Argonautas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Larva/metabolismo , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
We combine the labeling of newly transcribed RNAs with 5-ethynyluridine with the characterization of bound proteins. This approach, named capture of the newly transcribed RNA interactome using click chemistry (RICK), systematically captures proteins bound to a wide range of RNAs, including nascent RNAs and traditionally neglected nonpolyadenylated RNAs. RICK has identified mitotic regulators amongst other novel RNA-binding proteins with preferential affinity for nonpolyadenylated RNAs, revealed a link between metabolic enzymes/factors and nascent RNAs, and expanded the known RNA-bound proteome of mouse embryonic stem cells. RICK will facilitate an in-depth interrogation of the total RNA-bound proteome in different cells and systems.
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Química Click/métodos , Proteoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Espectrometria de Massas/métodos , Camundongos , Mapas de Interação de Proteínas , RNA/genética , Proteínas de Ligação a RNA/genética , Uridina/análogos & derivados , Uridina/químicaRESUMO
The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein-RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3' overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells.
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Proteínas Argonautas/metabolismo , Células-Tronco Embrionárias/enzimologia , MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoprecipitação , MicroRNAs/química , Clivagem do RNA , Precursores de RNA/química , RNA Mensageiro/metabolismo , Análise de Sequência de RNARESUMO
Endogenous small interfering RNAs (endo-siRNAs) are a newly emerged class of small regulatory RNAs. In Drosophila melanogaster, the production of endo-siRNAs depends on the RNase III enzyme Dicer-2 (Dcr-2). Loss of dcr-2 function reduces the resistance of adult flies to various stresses and shortens their life span. The mutants also exhibit alterations in carbohydrate and lipid metabolism. These findings suggest that the endo-siRNA pathway plays a protective role during exposure to stress and aging in D. melanogaster, possibly by regulating metabolic homeostasis. Here, we describe the methods that were used to discover the phenotypes associated with endo-siRNA deficiency by using dcr-2 null mutants.
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Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , RNA Helicases/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/genética , Animais , Temperatura Baixa , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/genética , Estresse do Retículo Endoplasmático , Deleção de Genes , Glucose/análise , Glucose/metabolismo , Hemolinfa/química , Hemolinfa/metabolismo , Longevidade , Estresse Oxidativo , RNA Helicases/metabolismo , Ribonuclease III/metabolismo , Estresse Fisiológico , Triglicerídeos/análise , Triglicerídeos/metabolismoRESUMO
The development of gene delivery systems into embryos is challenging due to technical difficulties, delivery efficiency and toxicity. Here, we developed an organic compound (VisuFect)-mediated gene delivery system for zygotes. The VisuFect, which is hydrophilic and Cy5.5-labeled, was conjugated with poly(A) oligo (VFA). The VFA into CHO cells showed clathrin-mediated internalization and no toxicity. The VFA successfully penetrated through the zona pellucida of fertilized eggs of various species including pigs, zebrafish, drosophilas and mice. The experiment with VisuFect-mediated delivery of the miR34c inhibitor showed similar results with direct microinjection of the miR34c inhibitor by suppressing the development of zygotes up to the blastocyst stage. Noticeable features of the VisuFect will provide great benefits for further studies on gene function in sperms and embryos.