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1.
Int J Mol Sci ; 23(16)2022 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-36012225

RESUMO

Regulation at the RNA level by RNA-binding proteins (RBPs) and microRNAs (miRNAs) is key to coordinating eukaryotic gene expression. In plants, the importance of miRNAs is highlighted by severe developmental defects in mutants impaired in miRNA biogenesis. MiRNAs are processed from long primary-microRNAs (pri-miRNAs) with internal stem-loop structures by endonucleolytic cleavage. The highly structured stem-loops constitute the basis for the extensive regulation of miRNA biogenesis through interaction with RBPs. However, trans-acting regulators of the biogenesis of specific miRNAs are largely unknown in plants. Therefore, we exploit an RNA-centric approach based on modified versions of the conditional CRISPR nuclease Csy4* to pull down interactors of the Arabidopsis pri-miR398b stem-loop (pri-miR398b-SL) in vitro. We designed three epitope-tagged versions of the inactive Csy4* for the immobilization of the protein together with the pri-miR398b-SL bait on high affinity matrices. After incubation with nucleoplasmic extracts from Arabidopsis and extensive washing, pri-miR398b-SL, along with its specifically bound proteins, were released by re-activating the cleavage activity of the Csy4* upon the addition of imidazole. Co-purified proteins were identified via quantitative mass spectrometry and data sets were compared. In total, we identified more than 400 different proteins, of which 180 are co-purified in at least two out of three independent Csy4*-based RNA pulldowns. Among those, the glycine-rich RNA-binding protein AtRZ-1a was identified in all pulldowns. To analyze the role of AtRZ-1a in miRNA biogenesis, we determined the miR398 expression level in the atrz-1a mutant. Indeed, the absence of AtRZ-1a caused a decrease in the steady-state level of mature miR398 with a concomitant reduction in pri-miR398b levels. Overall, we show that our modified Csy4*-based RNA pulldown strategy is suitable to identify new trans-acting regulators of miRNA biogenesis and provides new insights into the post-transcriptional regulation of miRNA processing by plant RBPs.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , MicroRNAs , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo
2.
Ecotoxicol Environ Saf ; 243: 113990, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35998476

RESUMO

Arsenic (As) is a natural hepatotoxicity inducer that is ubiquitous in water, soil, coal, and food. Studies have found that arsenite exposure elicits increased mRNA transcription and decreased protein expression of SOD1 in vivo and in vitro; however, the specific mechanisms remain unclear. Here, we established a model of arsenic-induced chronic liver injury by providing rats with drinking water containing different concentrations of sodium arsenite (NaAsO2) and found that NaAsO2 exposure decreased the mRNA and protein levels of AUF1 and the protein level of SOD1 and elevated the mRNA and protein levels of Dicer1 and miR-155 and the mRNA level of SOD1. Overexpression of AUF1 under NaAsO2 stress in vitro induced Dicer1 mRNA and protein expression and decreased miR-155 levels, which could be reversed by AUF1 siRNA. In addition, miR-155 overexpression downregulated SOD1 mRNA and protein levels, although this change was inhibited after transfection with an miR-155 inhibitor. Taken together, our findings showed that NaAsO2 could upregulate Dicer1 mRNA and protein, thereby increasing miR-155 expression by downregulating AUF1 mRNA and protein expression. A dual-luciferase reporter assay indicated that miR-155 decreased the mRNA and protein levels of SOD1 by targeting the SOD1 3'UTR, resulting in liver injury. This study provides an important research basis for further understanding the factors underlying arsenic-induced liver injury to improve the prevention and control strategies for arsenism.


Assuntos
Arsênio , Arsenitos , Doença Hepática Crônica Induzida por Substâncias e Drogas , Ribonucleoproteínas Nucleares Heterogêneas Grupo D , MicroRNAs , Regiões 3' não Traduzidas/genética , Animais , Arsênio/metabolismo , Arsênio/toxicidade , Arsenitos/metabolismo , Arsenitos/toxicidade , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ribonuclease III/genética , Ribonuclease III/metabolismo , Compostos de Sódio , Superóxido Dismutase-1/genética
3.
Nat Commun ; 13(1): 4663, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35945211

RESUMO

Kiss1 neurons, producing kisspeptins, are essential for puberty and fertility, but their molecular regulatory mechanisms remain unfolded. Here, we report that congenital ablation of the microRNA-synthesizing enzyme, Dicer, in Kiss1 cells, causes late-onset hypogonadotropic hypogonadism in both sexes, but is compatible with pubertal initiation and preserved Kiss1 neuronal populations at the infantile/juvenile period. Yet, failure to complete puberty and attain fertility is observed only in females. Kiss1-specific ablation of Dicer evokes disparate changes of Kiss1-cell numbers and Kiss1/kisspeptin expression between hypothalamic subpopulations during the pubertal-transition, with a predominant decline in arcuate-nucleus Kiss1 levels, linked to enhanced expression of its repressors, Mkrn3, Cbx7 and Eap1. Our data unveil that miRNA-biosynthesis in Kiss1 neurons is essential for pubertal completion and fertility, especially in females, but dispensable for initial reproductive maturation and neuronal survival in both sexes. Our results disclose a predominant miRNA-mediated inhibitory program of repressive signals that is key for precise regulation of Kiss1 expression and, thereby, reproductive function.


Assuntos
RNA Helicases DEAD-box/metabolismo , Kisspeptinas , Ribonuclease III/metabolismo , Animais , Feminino , Fertilidade , Kisspeptinas/genética , Kisspeptinas/metabolismo , Masculino , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , Ribonuclease III/genética , Maturidade Sexual/genética
4.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 30(4): 1286-1290, 2022 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-35981400

RESUMO

Myelodysplastic syndrome (MDS) are a heterogeneous group of hematological malignancies. Currently, in addition to demethylated chemotherapy and hematopoietic stem cell transplantation, MDS patient-derived mesenchymal stem cells (MDS-MSC) play an important role in understanding the pathogenesis of MDS and related therapeutic targets. For example, abnormal expression of DICER1 gene, abnormalities of PI3K/AKT and Wnt/ß-catenin signaling pathways provide new therapeutic targets for MDS. In addition, MDS-MSC is also affected by abnormal microenvironment of the body, such as inflammatory factor S100A9, as well as hypercoagulation and iron overload. In this review, genes, signaling pathways, cytokines, hematopoietic microenvironment, and the effect of therapeutic drugs for MDS-MSC were briefly summarized.


Assuntos
Neoplasias Hematológicas , Células-Tronco Mesenquimais , Síndromes Mielodisplásicas , Citocinas/metabolismo , RNA Helicases DEAD-box/metabolismo , Neoplasias Hematológicas/metabolismo , Humanos , Síndromes Mielodisplásicas/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ribonuclease III/metabolismo , Microambiente Tumoral
5.
Cell Mol Life Sci ; 79(8): 446, 2022 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-35876890

RESUMO

Increasing evidence suggests different, not completely understood roles of microRNA biogenesis in the development and progression of lung cancer. The overexpression of the DNA repair protein apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an important cause of poor chemotherapeutic response in lung cancer and its involvement in onco-miRNAs biogenesis has been recently described. Whether APE1 regulates miRNAs acting as prognostic biomarkers of lung cancer has not been investigated, yet. In this study, we analyzed miRNAs differential expression upon APE1 depletion in the A549 lung cancer cell line using high-throughput methods. We defined a signature of 13 miRNAs that strongly correlate with APE1 expression in human lung cancer: miR-1246, miR-4488, miR-24, miR-183, miR-660, miR-130b, miR-543, miR-200c, miR-376c, miR-218, miR-146a, miR-92b and miR-33a. Functional enrichment analysis of this signature revealed its biological relevance in cancer cell proliferation and survival. We validated DICER1 as a direct functional target of the APE1-regulated miRNA-33a-5p and miR-130b-3p. Importantly, IHC analyses of different human tumors confirmed a negative correlation existing between APE1 and Dicer1 protein levels. DICER1 downregulation represents a prognostic marker of cancer development but the mechanisms at the basis of this phenomenon are still completely unknown. Our findings, suggesting that APE1 modulates DICER1 expression via miR-33a and miR-130b, reveal new mechanistic insights on DICER1 regulation, which are of relevance in lung cancer chemoresistance and cancer invasiveness.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo
6.
Nucleic Acids Res ; 50(13): 7637-7654, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35801921

RESUMO

Although the route to generate microRNAs (miRNAs) is often depicted as a linear series of sequential and constitutive cleavages, we now appreciate multiple alternative pathways as well as diverse strategies to modulate their processing and function. Here, we identify an unusually profound regulatory role of conserved loop sequences in vertebrate pre-mir-144, which are essential for its cleavage by the Dicer RNase III enzyme in human and zebrafish models. Our data indicate that pre-mir-144 dicing is positively regulated via its terminal loop, and involves the ILF3 complex (NF90 and its partner NF45/ILF2). We provide further evidence that this regulatory switch involves reshaping of the pre-mir-144 apical loop into a structure that is appropriate for Dicer cleavage. In light of our recent findings that mir-144 promotes the nuclear biogenesis of its neighbor mir-451, these data extend the complex hierarchy of nuclear and cytoplasmic regulatory events that can control the maturation of clustered miRNAs.


Assuntos
MicroRNAs/genética , Ribonuclease III/metabolismo , Peixe-Zebra , Animais , Humanos , MicroRNAs/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
7.
RNA Biol ; 19(1): 908-915, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35829618

RESUMO

The RNase III family of dsRNA-specific endonucleases is exemplified by prokaryotic RNase III and eukaryotic Rnt1p, Drosha, and Dicer. Structures of Aquifex aeolicus RNase III (AaRNase III) and Saccharomyces cerevisiae Rnt1p (ScRnt1p) show that both enzymes recognize substrates in a sequence-specific manner and propel RNA hydrolysis by two-Mg2+-ion catalysis. Previously, we created an Escherichia coli RNase III variant (EcEEQ) by eliminating the sequence specificity via protein engineering and called it bacterial Dicer for the fact that it produces heterogeneous small interfering RNA cocktails. Here, we present a 1.8-Å crystal structure of a postcleavage complex of EcEEQ, representing a reaction state immediately after the cleavage of scissile bond. The structure not only establishes the structure-and-function relationship of EcEEQ, but also reveals the functional role of a third Mg2+ ion that is involved in RNA hydrolysis by bacterial RNase III. In contrast, the cleavage site assembly of ScRnt1p does not contain a third Mg2+ ion. Instead, it involves two more amino acid side chains conserved among eukaryotic RNase IIIs. We conclude that the EcEEQ structure (this work) represents the cleavage assembly of prokaryotic RNase IIIs and the ScRnt1p structure (PDB: 4OOG), also determined at the postcleavage state, represents the cleavage assembly of eukaryotic RNase IIIs. Together, these two structures provide insights into the reaction trajectory of two-Mg2+-ion catalysis by prokaryotic and eukaryotic RNase III enzymes.


Assuntos
Magnésio/metabolismo , Ribonuclease III , Proteínas de Saccharomyces cerevisiae , Biocatálise , Catálise , RNA de Cadeia Dupla , Ribonuclease III/genética , Ribonuclease III/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
8.
PLoS Genet ; 18(6): e1010267, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35714159

RESUMO

The conserved nucleic acid binding protein Translin contributes to numerous facets of mammalian biology and genetic diseases. It was first identified as a binder of cancer-associated chromosomal translocation breakpoint junctions leading to the suggestion that it was involved in genetic recombination. With a paralogous partner protein, Trax, Translin has subsequently been found to form a hetero-octomeric RNase complex that drives some of its functions, including passenger strand removal in RNA interference (RNAi). The Translin-Trax complex also degrades the precursors to tumour suppressing microRNAs in cancers deficient for the RNase III Dicer. This oncogenic activity has resulted in the Translin-Trax complex being explored as a therapeutic target. Additionally, Translin and Trax have been implicated in a wider range of biological functions ranging from sleep regulation to telomere transcript control. Here we reveal a Trax- and RNAi-independent function for Translin in dissociating RNA polymerase II from its genomic template, with loss of Translin function resulting in increased transcription-associated recombination and elevated genome instability. This provides genetic insight into the longstanding question of how Translin might influence chromosomal rearrangements in human genetic diseases and provides important functional understanding of an oncological therapeutic target.


Assuntos
RNA Polimerase II , Ribonuclease III , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica/genética , Humanos , Mamíferos/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo
9.
Nat Commun ; 13(1): 3560, 2022 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-35732654

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Pequeno RNA não Traduzido , Ribonuclease III , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo
10.
Mol Microbiol ; 118(1-2): 30-46, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35652235

RESUMO

Despite significant progress in understanding the diversity of tRNA processing pathways in Escherichia coli, the mechanism for the maturation of tRNAs encoded within the rRNA operons has not received much attention. Here, we show that the Glu2, Ile1, and Ala1B tRNAs, encoded by 10 genes located between the 16S and 23S rRNAs in the seven rRNA operons, are matured via a RNase E-independent processing pathway that utilizes at least six different enzymes. It has been shown that the Glu2 and Ile1-Ala1B pre-tRNAs released by initial RNase III cleavages of the 30S primary rRNA transcripts retain extended 5'-leader (35-139 nt) and 3'-trailer (166-185 nt) sequences. However, the 5' maturation of the tRNAs by RNase P is inhibited until the trailer sequences are shortened to 1-4 nucleotides, initially by a second RNase III cleavage at 31-42 nucleotides downstream of the CCA determinant followed by exonucleolytic trimming. The RNase III cleaved Glu2 and Ile1-Ala1B trailer fragments are degraded via PAP I- dependent exonucleolytic decay. Compared to the six previously characterized tRNA processing pathways, maturation of the Glu2, Ile1, and Ala1B tRNAs is considerably more complex and appears to be distinct from what occurs in Gram-positive bacteria.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Nucleotídeos/metabolismo , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribonuclease III/metabolismo , Ribonuclease P/genética , Ribonuclease P/metabolismo
11.
Nature ; 607(7918): 393-398, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35768503

RESUMO

In flies, Argonaute2 (Ago2) and small interfering RNA (siRNA) form an RNA-induced silencing complex to repress viral transcripts1. The RNase III enzyme Dicer-2 associates with its partner protein R2D2 and cleaves long double-stranded RNAs to produce 21-nucleotide siRNA duplexes, which are then loaded into Ago2 in a defined orientation2-5. Here we report cryo-electron microscopy structures of the Dicer-2-R2D2 and Dicer-2-R2D2-siRNA complexes. R2D2 interacts with the helicase domain and the central linker of Dicer-2 to inhibit the promiscuous processing of microRNA precursors by Dicer-2. Notably, our structure represents the strand-selection state in the siRNA-loading process, and reveals that R2D2 asymmetrically recognizes the end of the siRNA duplex with the higher base-pairing stability, and the other end is exposed to the solvent and is accessible by Ago2. Our findings explain how R2D2 senses the thermodynamic asymmetry of the siRNA and facilitates the siRNA loading into Ago2 in a defined orientation, thereby determining which strand of the siRNA duplex is used by Ago2 as the guide strand for target silencing.


Assuntos
Microscopia Crioeletrônica , Proteínas de Drosophila , RNA Helicases , RNA de Cadeia Dupla , RNA Interferente Pequeno , Proteínas de Ligação a RNA , Ribonuclease III , Animais , Proteínas Argonauta/metabolismo , Pareamento de Bases , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , MicroRNAs/metabolismo , Multimerização Proteica , RNA Helicases/química , RNA Helicases/metabolismo , RNA Helicases/ultraestrutura , Interferência de RNA , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/ultraestrutura , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/ultraestrutura , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Complexo de Inativação Induzido por RNA/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Ribonuclease III/ultraestrutura
12.
Nature ; 607(7918): 399-406, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35768513

RESUMO

Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes1,2. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs)3,4. ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes5,6. Here we report the cryo-electron microscopy structures of Dcr-2-Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5'-phosphate-binding pocket. The overall conformation of Dcr-2-Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2-Loqs-PD.


Assuntos
Microscopia Crioeletrônica , Proteínas de Drosophila , Drosophila melanogaster , RNA Helicases , RNA de Cadeia Dupla , RNA Interferente Pequeno , Proteínas de Ligação a RNA , Ribonuclease III , Trifosfato de Adenosina , Animais , Sítios de Ligação , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/ultraestrutura , Fosfatos/metabolismo , Conformação Proteica , RNA Helicases/química , RNA Helicases/metabolismo , RNA Helicases/ultraestrutura , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/ultraestrutura , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/ultraestrutura , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Ribonuclease III/química , Ribonuclease III/metabolismo , Ribonuclease III/ultraestrutura
13.
Nat Commun ; 13(1): 3558, 2022 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-35732665

RESUMO

Treatment of methicillin-resistant Staphylococcus aureus infections is dependent on the efficacy of last-line antibiotics including vancomycin. Treatment failure is commonly linked to isolates with intermediate vancomycin resistance (termed VISA). These isolates have accumulated point mutations that collectively reduce vancomycin sensitivity, often by thickening the cell wall. Changes in regulatory small RNA expression have been correlated with antibiotic stress in VISA isolates however the functions of most RNA regulators is unknown. Here we capture RNA-RNA interactions associated with RNase III using CLASH. RNase III-CLASH uncovers hundreds of novel RNA-RNA interactions in vivo allowing functional characterisation of many sRNAs for the first time. Surprisingly, many mRNA-mRNA interactions are recovered and we find that an mRNA encoding a long 3' untranslated region (UTR) (termed vigR 3'UTR) functions as a regulatory 'hub' within the RNA-RNA interaction network. We demonstrate that the vigR 3'UTR promotes expression of folD and the cell wall lytic transglycosylase isaA through direct mRNA-mRNA base-pairing. Deletion of the vigR 3'UTR re-sensitised VISA to glycopeptide treatment and both isaA and vigR 3'UTR deletions impact cell wall thickness. Our results demonstrate the utility of RNase III-CLASH and indicate that S. aureus uses mRNA-mRNA interactions to co-ordinate gene expression more widely than previously appreciated.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Ribonuclease III , Resistência a Vancomicina , Regiões 3' não Traduzidas/genética , Antibacterianos/uso terapêutico , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Vancomicina/farmacologia , Resistência a Vancomicina/genética
14.
Hum Mol Genet ; 31(17): 2934-2950, 2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-35405010

RESUMO

DROSHA encodes a ribonuclease that is a subunit of the Microprocessor complex and is involved in the first step of microRNA (miRNA) biogenesis. To date, DROSHA has not yet been associated with a Mendelian disease. Here, we describe two individuals with profound intellectual disability, epilepsy, white matter atrophy, microcephaly and dysmorphic features, who carry damaging de novo heterozygous variants in DROSHA. DROSHA is constrained for missense variants and moderately intolerant to loss-of-function (o/e = 0.24). The loss of the fruit fly ortholog drosha causes developmental arrest and death in third instar larvae, a severe reduction in brain size and loss of imaginal discs in the larva. Loss of drosha in eye clones causes small and rough eyes in adult flies. One of the identified DROSHA variants (p.Asp1219Gly) behaves as a strong loss-of-function allele in flies, while another variant (p.Arg1342Trp) is less damaging in our assays. In worms, a knock-in that mimics the p.Asp1219Gly variant at a worm equivalent residue causes loss of miRNA expression and heterochronicity, a phenotype characteristic of the loss of miRNA. Together, our data show that the DROSHA variants found in the individuals presented here are damaging based on functional studies in model organisms and likely underlie the severe phenotype involving the nervous system.


Assuntos
Epilepsia , Deficiência Intelectual , MicroRNAs , Microcefalia , Malformações do Sistema Nervoso , Humanos , Deficiência Intelectual/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Microcefalia/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo
15.
Nucleic Acids Res ; 50(8): 4669-4684, 2022 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-35380679

RESUMO

Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt small interfering RNAs (siRNAs), heterochromatic siRNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs), respectively. The former small RNAs are linked to silencing of transposable elements and heterochromatic repeats, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient 'eudicot-type' DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 5' phosphates and 3' overhangs, consistent with the structural properties of their in vivo double-stranded RNA substrates. Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5, which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution.


Assuntos
Proteínas de Arabidopsis , Ribonuclease III , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Duplicação Gênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Interferência de RNA , RNA de Cadeia Dupla , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo
16.
Nat Commun ; 13(1): 2138, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35440644

RESUMO

The accurate and efficient cleavage of shRNAs and pre-miRNAs by DICER is crucial for their gene-silencing activity. Here, we conduct high-throughput DICER cleavage assays for more than ~20,000 different shRNAs and show the comprehensive cleavage activities of DICER on these sequences. We discover a single-nucleotide bulge (22-bulge), which facilitates the cleavage activity of DICER on shRNAs and human pre-miRNAs. As a result, this 22-bulge enhances the gene-silencing activity of shRNAs and the accuracy of miRNA biogenesis. In addition, various single-nucleotide polymorphism-edited 22-bulges are found to govern the cleavage sites of DICER on pre-miRNAs and thereby control their functions. Finally, we identify the single cleavage of DICER and reveal its molecular mechanism. Our findings improve the understanding of the DICER cleavage mechanism, provide a foundation for the design of accurate and efficient shRNAs for gene-silencing, and indicate the function of bulges in regulating miRNA biogenesis.


Assuntos
MicroRNAs , Precursores de RNA , Inativação Gênica , Humanos , MicroRNAs/química , MicroRNAs/genética , Nucleotídeos , Precursores de RNA/genética , RNA Interferente Pequeno/genética , Ribonuclease III/metabolismo
17.
J Tradit Chin Med ; 42(1): 108-115, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35294130

RESUMO

OBJECTIVE: To further clarify the anticancer mechanisms of Liujunzi decoction and provide possible targets for the treatment of advanced-stage nonsmall cell lung cancer (NSCLC) by re-analyzing differential gene expression profile of peripheral blood mononuclear cells (PBMCs) from Liujunzi decoctiontreated NSCLC patients receiving first-line chemotherapy. METHODS: The PBMC gene expression microarray data set GSE61926 was retrieved from a high throughput gene expression database. Differentially expressed genes (DEGs) were screened by paired sample t-test and the multiple ratio method. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were performed using the DAVID database. The protein-protein interaction (PPI) network was constructed using interaction gene library retrieval tools and Cytoscape software. RESULTS: A total of 162 DEGs were identified, with 67 upregulated genes and 95 downregulated genes. The functional distribution of Gene Oncology (GO) genes showed that DEGs were mostly concentrated in extracellular regions, calcium ion binding, and transcriptase activity. KEGG pathway analysis showed that cytokine-cytokine receptor interactions were significantly enriched. PPI network analysis screened out the top 10 central protein-coding genes with the highest nodal degree: IL2, PIWIL4, DICER1, PIWIL2, SAA1, XCL1, IL22RA1, ARHGAP11A, DCP1A, and GDNF. Among them, the central protein-coding gene with the highest node degree was IL2. In addition, the central protein-coding genes with high node degrees and high molecular complex detection (MCODE) scores were PIWIL4, DICER1, PIWIL2, and DCP1A, all of which are related to tumor development. CONCLUSIONS: One signaling pathway and 10 central protein-coding genes related to anticancer mechanisms were screened by re-analysis of GSE61926 data. IL2, PIWIL4, DICER1, PIWIL2, and DCP1A may have important roles in the mechanism of Liujunzi decoction treatment against NSCLC. Our results suggest that the anticancer mechanism of Liujunzi decoction may be related to gene silencing by RNA and the biological processes of piwi-interacting RNA and other small RNAs.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Biologia Computacional/métodos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Medicamentos de Ervas Chinesas , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-2/genética , Leucócitos Mononucleares/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo
18.
Biochem Biophys Res Commun ; 605: 97-103, 2022 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-35316769

RESUMO

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are crucial for plant growth and development via mediating post-transcriptional gene silencing. In wild-type Arabidopsis, DICER-LIKE 2 (DCL2)-dependent 22-nt siRNAs are rare, whereas DCL1 and DCL4-dependent 21-nt miRNAs and siRNAs are highly abundant. DCL4 naturally inhibits DCL2 in producing abundant 22-nt siRNAs from endogenous transcripts, but whether DCL1 suppresses endogenous 22-nt siRNA production and the extent of repression are still unknown. Here, we report that DCL1 and DCL2 cleaved both miRNA precursors and coding transcript-derived double-stranded RNAs. In a dcl1 dcl4 double mutant, massive 22-nt siRNAs were produced from endogenous protein-coding genes (genic siRNAs). Compared with wild-type, the 22-nt genic siRNAs derived from the Nitrate Reductase 1 (NIA1), NIA2, DIACYLGLYCEROL ACYLTRANSFERASES 3 (DGAT3), SUPPRESSOR OF MAX2 1-LIKE 5 (SMXL5), and SMXL4 in dcl1 dcl4 increased up to 95%. Our analysis further indicated that the 22-nt genic siRNAs in dcl1 dcl4 were mainly loaded into ARGONAUTE 1 (AGO1) or AGO2. Thus, our results demonstrated that both DCL1 and DCL4 safeguard post-transcriptional gene silencing, preventing the production of DCL2-dependent 22-nt genic siRNAs from disrupting plant growth and development.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , MicroRNAs , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , RNA de Cadeia Dupla , RNA Interferente Pequeno/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo
19.
J Exp Clin Cancer Res ; 41(1): 69, 2022 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-35183226

RESUMO

BACKGROUND: Glycolysis is a pivotal process in metabolic reprogramming of tumorigenesis. Previous research has indicated that lncRNAs might play crucial roles in glycolysis of various tumors. However, the function of lncRNAs in glycolysis of pancreatic cancer has not been fully elucidated. METHODS: Bio-information analyses were applied to reveal the potential glycolysis-associated lncRNA. RT-PCR and fluorescence in situ hybridization (FISH) assays were applied to detect the expression of antisense RNA1 of DICER1 (DICER1-AS1) in pancreatic cancer tissues and cell lines. Gain- and loss-of-function experiments were performed to evaluate the roles of DICER1-AS1 in glycolysis and tumorigenesis of PC. Mechanistic experiments including luciferase reporter assay, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP) were employed to uncover the downstream targets and regulatory mechanism of DICER1-AS1 in glycolysis of PC. RESULTS: Bio-information analysis indicated that DICER1-AS1 was downregulated in PC and negatively correlated with glycolytic genes expression. Meanwhile, overexpression of DICER1-AS1 inhibited glycolysis, proliferation, and metastasis of PC cells both in vitro and in vivo. Mechanistically, DICER1-AS1 promoted transcription of its sense gene DICER1 by recruiting transcriptional factor YY1 to the DICER1 promoter. Meanwhile, DICER1 promoted maturation of miR-5586-5p which consequently inhibited glycolytic gene expression including LDHA, HK2, PGK1, and SLC2A1. Notably, enhanced interaction between N6-methyladenosine (m6A) reader YTHDF3 and DICER1-AS1 led to degradation of DICER1-AS1 in response to glucose depletion. Moreover, our data revealed that YTHDF3 was a critical target for miR-5586-5p, by which forming a negative feedback with DICER1-AS1 to regulate glycolysis of PC. CONCLUSION: Our results implicate a negative feedback of m6A reader YTHDF3 and glycolytic lncRNA DICER1-AS1 is involved in glycolysis and tumorigenesis of PC.


Assuntos
Adenosina/análogos & derivados , RNA Helicases DEAD-box/metabolismo , Neoplasias Pancreáticas/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo , Adenosina/metabolismo , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Glicólise , Humanos , Neoplasias Pancreáticas/patologia , Transfecção
20.
Genes Cells ; 27(4): 280-292, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35143697

RESUMO

Dicer-like 1 (DCL1) is a core component of the plant microRNA (miRNA) biogenesis machinery. MiRNA is transcribed as a precursor RNA, termed primary miRNA (pri-miRNA), which is cleaved by DCL1 in two steps to generate miRNA/miRNA* duplex. Pri-miRNA is a single-stranded RNA that forms a hairpin structure with a number of unpaired bases, hereafter called mismatches, on its stem. In the present study, by using purified recombinant Arabidopsis DCL1, we presented evidence that mismatches on the stem of pri-miRNA are important for precise DCL1 cleavage. We showed that a mismatch at the loop-distal side of the end of miRNA/miRNA* duplex is important for efficient cleavage of pri-miRNA in vitro, as previously suggested in planta. On the contrary, mismatches distant from the miRNA/miRNA* duplex region are important for determining the cleavage position by DCL1. The purified DCL1 proteins cleaved mutant pri-miRNA variants without such mismatches at a position at which wild-type pri-miRNA variants are not usually cleaved, resulting in an increased accumulation of small RNA different from miRNA. Therefore, our results suggest that, in addition to the distance from the ssRNA-dsRNA junction, mismatches on the stem of pri-miRNA function as a determinant for precise processing of pri-miRNA by DCL1 in plants.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Ciclo Celular , MicroRNAs , Ribonuclease III , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Nucleotídeos/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease III/genética , Ribonuclease III/metabolismo
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