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1.
Nat Commun ; 10(1): 2375, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31147543

RESUMO

Human antigen R (HuR) is a member of the Hu family of RNA-binding proteins and is involved in many physiological processes. Obesity, as a worldwide healthcare problem, has attracted more and more attention. To investigate the role of adipose HuR, we generate adipose-specific HuR knockout (HuRAKO) mice. As compared with control mice, HuRAKO mice show obesity when induced with a high-fat diet, along with insulin resistance, glucose intolerance, hypercholesterolemia and increased inflammation in adipose tissue. The obesity of HuRAKO mice is attributed to adipocyte hypertrophy in white adipose tissue due to decreased expression of adipose triglyceride lipase (ATGL). HuR positively regulates ATGL expression by promoting the mRNA stability and translation of ATGL. Consistently, the expression of HuR in adipose tissue is reduced in obese humans. This study suggests that adipose HuR may be a critical regulator of ATGL expression and lipolysis and thereby controls obesity and metabolic syndrome.


Assuntos
Tecido Adiposo Branco/metabolismo , Proteína Semelhante a ELAV 1/genética , Intolerância à Glucose/genética , Hipercolesterolemia/genética , Resistência à Insulina/genética , Lipase/genética , Obesidade/genética , Adipócitos/patologia , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Tecido Adiposo Branco/imunologia , Animais , Crescimento Celular , Dieta Hiperlipídica , Proteína Semelhante a ELAV 1/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Intolerância à Glucose/metabolismo , Humanos , Hipercolesterolemia/metabolismo , Hipertrofia , Inflamação/imunologia , Lipase/metabolismo , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Knockout , Obesidade/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA/genética , Gordura Subcutânea/metabolismo
2.
Nat Cell Biol ; 21(5): 542-551, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31048766

RESUMO

A diverse catalog of long noncoding RNAs (lncRNAs), which lack protein-coding potential, are transcribed from the mammalian genome. They are emerging as important regulators in gene expression networks by controlling nuclear architecture and transcription in the nucleus and by modulating mRNA stability, translation and post-translational modifications in the cytoplasm. In this Review, we highlight recent progress in cellular functions of lncRNAs at the molecular level in mammalian cells.


Assuntos
Fenômenos Fisiológicos Celulares/genética , Genoma/genética , Biossíntese de Proteínas/genética , RNA Longo não Codificante/genética , Animais , Redes Reguladoras de Genes/genética , Humanos , Processamento de Proteína Pós-Traducional/genética , Estabilidade de RNA/genética
3.
Nat Commun ; 10(1): 1969, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036808

RESUMO

Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Here we report on the characterization of an intergenic lncRNA expressed in epithelial tissues which we termed EPR (Epithelial cell Program Regulator). EPR is rapidly downregulated by TGF-ß and its sustained expression largely reshapes the transcriptome, favors the acquisition of epithelial traits, and reduces cell proliferation in cultured mammary gland cells as well as in an animal model of orthotopic transplantation. EPR generates a small peptide that localizes at epithelial cell junctions but the RNA molecule per se accounts for the vast majority of EPR-induced gene expression changes. Mechanistically, EPR interacts with chromatin and regulates Cdkn1a gene expression by affecting both its transcription and mRNA decay through its association with SMAD3 and the mRNA decay-promoting factor KHSRP, respectively. We propose that EPR enables epithelial cells to control proliferation by modulating waves of gene expression in response to TGF-ß.


Assuntos
Estabilidade de RNA/genética , RNA Longo não Codificante/genética , Proteína Smad3/metabolismo , Transcriptoma/genética , Fator de Crescimento Transformador beta/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , RNA Longo não Codificante/efeitos dos fármacos
4.
Mol Cell ; 74(3): 494-507.e8, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30930054

RESUMO

N6-methyladenosine (m6A) is the most abundant internal modification in RNAs and plays regulatory roles in a variety of biological and physiological processes. Despite its important roles, the molecular mechanism underlying m6A-mediated gene regulation is poorly understood. Here, we show that m6A-containing RNAs are subject to endoribonucleolytic cleavage via YTHDF2 (m6A reader protein), HRSP12 (adaptor protein), and RNase P/MRP (endoribonucleases). We demonstrate that HRSP12 functions as an adaptor to bridge YTHDF2 and RNase P/MRP, eliciting rapid degradation of YTHDF2-bound RNAs. Transcriptome-wide analyses show that m6A RNAs that are preferentially targeted for endoribonucleolytic cleavage have an HRSP12-binding site and a RNase P/MRP-directed cleavage site upstream and downstream of the YTHDF2-binding site, respectively. We also find that a subset of m6A-containing circular RNAs associates with YTHDF2 in an HRSP12-dependent manner and is selectively downregulated by RNase P/MRP. Thus, our data expand the known functions of RNase P/MRP to endoribonucleolytic cleavage of m6A RNAs.


Assuntos
Adenosina/análogos & derivados , Proteínas de Choque Térmico/genética , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Ribonuclease P/genética , Ribonucleases/genética , Adenosina/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Sítios de Ligação/genética , Escherichia coli/genética , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , Metiltransferases/genética , RNA/genética , Processamento Pós-Transcricional do RNA/genética , Transcriptoma/genética
5.
Biomed Res Int ; 2019: 7398208, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30941371

RESUMO

Ribonuclease L (RNase L) is an important antiviral endoribonuclease regulated by type I IFN. RNase L is activated by viral infection and dsRNA. Because the role of swine RNase L (sRNase L) is not fully understood, in this study, we generated a sRNase L knockout PK-15 (KO-PK) cell line through the CRISPR/Cas9 gene editing system to evaluate the function of sRNase L. After transfection with CRISPR-Cas9 followed by selection using puromycin, sRNase L knockout in PK-15 cells was further validated by agarose gel electrophoresis, DNA sequencing, and Western blotting. The sRNase L KO-PK cells failed to trigger RNA degradation and induced less apoptosis than the parental PK-15 cells after transfected with poly (I: C). Furthermore, the levels of ISGs mRNA in sRNase L KO-PK cells were higher than those in the parental PK-15 cells after treated with poly (I: C). Finally, both wild type and attenuated pseudorabies viruses (PRV) replicated more efficiently in sRNase L KO-PK cells than the parental PK-15 cells. Taken together, these findings suggest that sRNase L has multiple biological functions including cellular single-stranded RNA degradation, induction of apoptosis, downregulation of transcript levels of ISGs, and antiviral activity against PRV. The sRNase L KO-PK cell line will be a valuable tool for studying functions of sRNase L as well as for producing PRV attenuated vaccine.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Endorribonucleases/metabolismo , Técnicas de Inativação de Genes , Herpesvirus Suídeo 1/fisiologia , Replicação Viral/fisiologia , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Linhagem Celular , Edição de Genes , Herpesvirus Suídeo 1/efeitos dos fármacos , Herpesvirus Suídeo 1/crescimento & desenvolvimento , Poli I-C/farmacologia , Pseudorraiva/virologia , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico/genética , Suínos , Vacinas Virais/imunologia , Replicação Viral/efeitos dos fármacos
6.
Nat Protoc ; 14(5): 1603-1633, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31019309

RESUMO

RNA degradation ensures appropriate levels of mRNA transcripts within cells and eliminates aberrant RNAs. Detailed studies of RNA degradation dynamics have been heretofore infeasible because of the inherent instability of degradation intermediates due to the high processivity of the enzymes involved. To visualize decay intermediates and to characterize the spatiotemporal dynamics of mRNA decay, we have developed a set of methods that apply XRN1-resistant RNA sequences (xrRNAs) to protect mRNA transcripts from 5'-3' exonucleolytic digestion. To our knowledge, this approach is the only method that can detect the directionality of mRNA degradation and that allows tracking of degradation products in unperturbed cells. Here, we provide detailed procedures for xrRNA reporter design, transfection and cell line generation. We explain how to extract xrRNA reporter mRNAs from mammalian cells, as well as their detection and quantification using northern blotting and quantitative PCR. The procedure further focuses on how to detect and quantify intact reporter mRNAs and XRN1-resistant degradation intermediates using single-molecule fluorescence microscopy. It provides detailed instructions for sample preparation and image acquisition using fixed, as well as living, cells. The procedure puts special emphasis on detailed descriptions of high-throughput image analysis pipelines, which are provided along with the article and were designed to perform spot co-localization, detection efficiency normalization and the quality control steps necessary for interpretation of results. The aim of the analysis software published here is to enable nonexpert readers to detect and quantify RNA decay intermediates within 4-6 d after reporter mRNA expression.


Assuntos
Estabilidade de RNA/genética , RNA Mensageiro/análise , RNA Mensageiro/química , Imagem Individual de Molécula/métodos , Exorribonucleases , Microscopia de Fluorescência , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Genes Dev ; 33(9-10): 536-549, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30842217

RESUMO

The exosome functions in the degradation of diverse RNA species, yet how it is negatively regulated remains largely unknown. Here, we show that NRDE2 forms a 1:1 complex with MTR4, a nuclear exosome cofactor critical for exosome recruitment, via a conserved MTR4-interacting domain (MID). Unexpectedly, NRDE2 mainly localizes in nuclear speckles, where it inhibits MTR4 recruitment and RNA degradation, and thereby ensures efficient mRNA nuclear export. Structural and biochemical data revealed that NRDE2 interacts with MTR4's key residues, locks MTR4 in a closed conformation, and inhibits MTR4 interaction with the exosome as well as proteins important for MTR4 recruitment, such as the cap-binding complex (CBC) and ZFC3H1. Functionally, MID deletion results in the loss of self-renewal of mouse embryonic stem cells. Together, our data pinpoint NRDE2 as a nuclear exosome negative regulator that ensures mRNA stability and nuclear export.


Assuntos
Exossomos/genética , Exossomos/metabolismo , Proteínas Nucleares/fisiologia , RNA Helicases/metabolismo , Animais , Núcleo Celular/metabolismo , Células-Tronco Embrionárias , Células HEK293 , Células HeLa , Humanos , Camundongos , Proteínas Nucleares/genética , Ligação Proteica , Domínios Proteicos , Transporte Proteico/genética , Estabilidade de RNA/genética
8.
Mol Cell ; 74(2): 284-295.e5, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30852060

RESUMO

The diversity of mRNA lifetimes in bacterial cells is difficult to reconcile with the relaxed cleavage site specificity of RNase E, the endonuclease most important for governing mRNA degradation. This enzyme has generally been thought to locate cleavage sites by searching freely in three dimensions. However, our results now show that its access to such sites in 5'-monophosphorylated RNA is hindered by obstacles-such as bound proteins or ribosomes or coaxial small RNA (sRNA) base pairing-that disrupt the path from the 5' end to those sites and prolong mRNA lifetimes. These findings suggest that RNase E searches for cleavage sites by scanning linearly from the 5'-terminal monophosphate along single-stranded regions of RNA and that its progress is impeded by structural discontinuities encountered along the way. This discovery has major implications for gene regulation in bacteria and suggests a general mechanism by which other prokaryotic and eukaryotic regulatory proteins can be controlled.


Assuntos
Endorribonucleases/genética , Escherichia coli/genética , Estabilidade de RNA/genética , RNA Bacteriano/genética , Endorribonucleases/química , Regulação Bacteriana da Expressão Gênica/genética , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Mensageiro/genética , Ribossomos/química , Ribossomos/genética
9.
J Exp Clin Cancer Res ; 38(1): 121, 2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850014

RESUMO

The p38 mitogen-activated protein kinase (p38MAPK) pathway has been implicated in a variety of pathological conditions including inflammation and metastasis. Post-transcriptional regulation of genes harboring adenine/uridine-rich elements (AREs) in their 3'-untranslated region (3'-UTR) is controlled by MAPK-activated protein kinase 2 (MAPKAPK2 or MK2), a downstream substrate of the p38MAPK. In response to diverse extracellular stimuli, MK2 influences crucial signaling events, regulates inflammatory cytokines, transcript stability and critical cellular processes. Expression of genes involved in these vital cellular cascades is controlled by subtle interactions in underlying molecular networks and post-transcriptional gene regulation that determines transcript fate in association with RNA-binding proteins (RBPs). Several RBPs associate with the 3'-UTRs of the target transcripts and regulate their expression via modulation of transcript stability. Although MK2 regulates important cellular phenomenon, yet its biological significance in tumor progression has not been well elucidated till date. In this review, we have highlighted in detail the importance of MK2 as the master regulator of RBPs and its role in the regulation of transcript stability, tumor progression, as well as the possibility of use of MK2 as a therapeutic target in tumor management.


Assuntos
Carcinogênese/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ligação a RNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/patologia , Estabilidade de RNA/genética , Transdução de Sinais/genética
10.
PLoS Pathog ; 15(2): e1007596, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30785952

RESUMO

Nuclear RNAs are subject to a number of RNA decay pathways that serve quality control and regulatory functions. As a result, any virus that expresses its genes in the nucleus must have evolved mechanisms that avoid these pathways, but the how viruses evade nuclear RNA decay remains largely unknown. The multifunctional Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 (Mta) protein is required for the nuclear stability of viral transcripts. In the absence of ORF57, we show that viral transcripts are subject to degradation by two specific nuclear RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) in which decay factors are recruited through poly(A) tails, and an ARS2-mediated RNA decay pathway dependent on the 5' RNA cap. In transcription pulse chase assays, ORF57 appears to act primarily by inhibiting the ARS2-mediated RNA decay pathway. In the context of viral infection in cultured cells, inactivation of both decay pathways by RNAi is necessary for the restoration of ORF57-dependent viral genes produced from an ORF57-null bacmid. Mechanistically, we demonstrate that ORF57 protects viral transcripts by preventing the recruitment of the exosome co-factor hMTR4. In addition, our data suggest that ORF57 recruitment of ALYREF inhibits hMTR4 association with some viral RNAs, whereas other KSHV transcripts are stabilized by ORF57 in an ALYREF-independent fashion. In conclusion, our studies show that KSHV RNAs are subject to nuclear degradation by two specific host pathways, PPD and ARS2-mediated decay, and ORF57 protects viral transcripts from decay by inhibiting hMTR4 recruitment.


Assuntos
RNA Helicases/metabolismo , Estabilidade de RNA/fisiologia , Proteínas Virais Reguladoras e Acessórias/metabolismo , Linhagem Celular , Núcleo Celular , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Regulação Viral da Expressão Gênica/genética , Genes Virais/genética , Células HEK293 , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/patogenicidade , Humanos , Proteínas Nucleares , Proteína I de Ligação a Poli(A)/metabolismo , Proteína I de Ligação a Poli(A)/fisiologia , Ligação Proteica , RNA Helicases/fisiologia , Estabilidade de RNA/genética , RNA Nuclear/fisiologia , RNA Viral , Proteínas de Ligação a RNA , Fatores de Transcrição , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/fisiologia , Replicação Viral
11.
Nat Commun ; 10(1): 563, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718516

RESUMO

Ribosome-associated quality control (RQC) pathways monitor and respond to ribosome stalling. Using in vivo UV-crosslinking and mass spectrometry, we identified a C-terminal region in Hel2/Rqt1 as an RNA binding domain. Complementary crosslinking and sequencing data for Hel2 revealed binding to 18S rRNA and translated mRNAs. Hel2 preferentially bound mRNAs upstream and downstream of the stop codon. C-terminal truncation of Hel2 abolished the major 18S crosslink and polysome association, and altered mRNA binding. HEL2 deletion caused loss of RQC and, we report here, no-go decay (NGD), with comparable effects for Hel2 truncation including the RNA-binding site. Asc1 acts upstream of Hel2 in RQC and asc1∆ impaired Hel2 binding to 18S and mRNA. In conclusion: Hel2 is recruited or stabilized on translating 40S ribosomal subunits by interactions with 18S rRNA and Asc1. This 18S interaction is required for Hel2 function in RQC and NGD. Hel2 probably interacts with mRNA during translation termination.


Assuntos
RNA Ribossômico 18S/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Mutação/genética , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética
12.
Nucleic Acids Res ; 47(6): 3045-3057, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30715470

RESUMO

Uridylation-dependent RNA decay is a widespread eukaryotic pathway modulating RNA homeostasis. Terminal uridylyltransferases (Tutases) add untemplated uridyl residues to RNA 3'-ends, marking them for degradation by the U-specific exonuclease Dis3L2. In Schizosaccharomyces pombe, Cid1 uridylates a variety of RNAs. In this study, we investigate the prevalence and impact of uridylation-dependent RNA decay in S. pombe by transcriptionally profiling cid1 and dis3L2 deletion strains. We found that the exonuclease Dis3L2 represents a bottleneck in uridylation-dependent mRNA decay, whereas Cid1 plays a redundant role that can be complemented by other Tutases. Deletion of dis3L2 elicits a cellular stress response, upregulating transcription of genes involved in protein folding and degradation. Misfolded proteins accumulate in both deletion strains, yet only trigger a strong stress response in dis3L2 deficient cells. While a deletion of cid1 increases sensitivity to protein misfolding stress, a dis3L2 deletion showed no increased sensitivity or was even protective. We furthermore show that uridylyl- and adenylyltransferases cooperate to generate a 5'-NxAUUAAAA-3' RNA motif on dak2 mRNA. Our studies elucidate the role of uridylation-dependent RNA decay as part of a global mRNA surveillance, and we found that perturbation of this pathway leads to the accumulation of misfolded proteins and elicits cellular stress responses.


Assuntos
RNA Nucleotidiltransferases/genética , Estabilidade de RNA/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Nucleotidiltransferases/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Uridina/genética
13.
Int J Mol Med ; 43(4): 1597-1610, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30720050

RESUMO

The current study aimed to clarify the role of nucleolin in the phenotypic transformation of vascular smooth muscle cells (VSMCs) and to preliminarily explore its underlying mechanism. The spatial and temporal expression patterns of nucleolin, and the effects of angiotensin II (Ang II) on the expression of VSMC phenotypic transformation markers, α­smooth muscle­actin, calponin, smooth muscle protein 22α and osteopontin were investigated. The effects of nucleolin on VSMC phenotypic transformation and the expression of phenotypic transformation­associated genes, tropoelastin, epiregulin and fibroblast growth factor 2 (b­FGF), were determined. Protein­RNA co­immunoprecipitation was used to investigate the potential target genes regulated by the nucleolin in phenotypic transformation of VSMCs. Finally, the stability of tropoelastin mRNA and the effects of nucleolin on the expression of tropoelastin were assayed. The results revealed that Ang II significantly promoted the phenotypic transformation of VSMCs. The expression of nucleolin was gradually upregulated in VSMCs treated with Ang II at different concentrations for various durations. Ang II induced nucleolin translocation from the nucleus to cytoplasm. Additionally, Ang II significantly promoted the phenotypic transformation of VSMCs. Overexpression and silencing of nucleolin regulated the expressions of tropoelastin, epiregulin and b­FGF. There was an interaction between tropoelastin mRNA and nucleolin protein, promoting the stability of tropoelastin mRNA and enhancing the expression of tropoelastin at the protein level. Upregulation of nucleolin had an important role in Ang II­induced VSMC phenotypic transformation, and its underlying mechanism may be through interacting with tropoelastin mRNA, leading to its increased stability and protein expression. The findings provide a new perspective into the regulatory mechanism of VSMC phenotypic transformation.


Assuntos
Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Tropoelastina/metabolismo , Angiotensina II , Animais , Linhagem Celular Transformada , Epirregulina/genética , Epirregulina/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Inativação Gênica , Fenótipo , Ligação Proteica , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Tropoelastina/genética
14.
Methods Mol Biol ; 1932: 197-213, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30701502

RESUMO

Degradome sequencing provides large amounts of data regarding RNA degradation. The degradome library construction described here is modified from the 5'-rapid amplification of cDNA ends (5'-RACE), and each degradome cDNA is sequenced by next-generation sequencing (NGS). Degradome profiles provide information confirming miRNA-mediated cleavage of target genes and allow the identification of novel targets. Furthermore, degradome sequencing provides additional information for the study of RNA processing, such as information regarding RNA-binding proteins. In this chapter, we describe a detailed optimized protocol for constructing a degradome library with high yield and quality, along with NGS and data mining procedures. We hope that the degradome approach will be applied to further studies of non-model organisms.


Assuntos
Plantas/genética , Estabilidade de RNA/genética , RNA de Plantas/genética , Análise de Sequência de RNA/métodos , Sequência de Bases , DNA Complementar/genética , Regulação da Expressão Gênica de Plantas/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Proteínas de Plantas/genética , Proteínas de Ligação a RNA/genética
15.
Methods Mol Biol ; 1932: 285-293, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30701508

RESUMO

Small noncoding RNAs of 20-30 nucleotides in length are key mediators of gene silencing. 2'-O-Methylation on the 3' terminal nucleotide of several types of small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants, PIWI-interacting RNAs (piRNAs) in animals, and some siRNAs in Drosophila and Caenorhabditis elegans, provides a key protective mechanism against 3' tailing- and trimming-mediated destabilization. The methylation reaction is catalyzed by the small RNA methyltransferase HUA ENHANCER 1 (HEN1). In this chapter, we describe a detailed protocol for analyzing 3' end methylation status of plant miRNAs, which can be applicable to other types of small RNAs as well.


Assuntos
Metiltransferases/genética , MicroRNAs/genética , RNA de Plantas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Metilação , Estabilidade de RNA/genética , RNA Interferente Pequeno/genética , Pequeno RNA não Traduzido/genética
16.
Cell Mol Gastroenterol Hepatol ; 7(2): 339-355, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30704983

RESUMO

BACKGROUND & AIMS: Hypoxia-associated pathways influence the development of inflammatory bowel disease. Adaptive responses to hypoxia are mediated through hypoxia-inducible factors, which are regulated by iron-dependent hydroxylases. Signals reflecting oxygen tension and iron levels in enterocytes regulate iron metabolism. Conversely, iron availability modulates responses to hypoxia. In the present study we sought to elucidate how iron influences the responses to hypoxia in the intestinal epithelium. METHODS: Human subjects were exposed to hypoxia, and colonic biopsy specimens and serum samples were collected. HT-29, Caco-2, and T84 cells were subjected to normoxia or hypoxia in the presence of iron or the iron chelator deferoxamine. Changes in inflammatory gene expression and signaling were assessed by quantitative polymerase chain reaction and Western blot. Chromatin immunoprecipitation was performed using antibodies against nuclear factor (NF)-κB and primers for the promoter of tumor necrosis factor (TNF) and interleukin (IL)1ß. RESULTS: Human subjects presented reduced levels of ferritin in the intestinal epithelium after hypoxia. Hypoxia reduced iron deprivation-associated TNF and IL1ß expression in HT-29 cells through the induction of autophagy. Contrarily, hypoxia triggered TNF and IL1ß expression, and NF-κB activation in Caco-2 and T84 cells. Iron blocked autophagy in Caco-2 cells, while reducing hypoxia-associated TNF and IL1ß expression through the inhibition of NF-κB binding to the promoter of TNF and IL1ß. CONCLUSIONS: Hypoxia promotes iron mobilization from the intestinal epithelium. Hypoxia-associated autophagy reduces inflammatory processes in HT-29 cells. In Caco-2 cells, iron uptake is essential to counteract hypoxia-induced inflammation. Iron mobilization into enterocytes may be a vital protective mechanism in the hypoxic inflamed mucosa.


Assuntos
Hipóxia/complicações , Inflamação/tratamento farmacológico , Inflamação/etiologia , Mucosa Intestinal/metabolismo , Ferro/uso terapêutico , NF-kappa B/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Autofagia/efeitos dos fármacos , Células CACO-2 , Células HT29 , Humanos , Inflamação/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Pessoa de Meia-Idade , Modelos Biológicos , Regiões Promotoras Genéticas/genética , Estabilidade de RNA/efeitos dos fármacos , Estabilidade de RNA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Adulto Jovem
17.
Biochim Biophys Acta Mol Cell Res ; 1866(3): 360-370, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30595162

RESUMO

Ras-GTPase-activating protein (SH3 domain)-binding proteins (G3BPs, also known as Rasputin) are a family of RNA binding proteins that regulate gene expression in response to environmental stresses by controlling mRNA stability and translation. G3BPs appear to facilitate this activity through their role in stress granules for which they are considered a core component, however, it should be noted that not all stress granules contain G3BPs and this appears to be contextual depending on the environmental stress and the cell type. Although the role of G3BPs in stress granules appears to be one of its major roles, data also strongly suggests that they interact with mRNAs outside of stress granules to regulate gene expression. G3BPs have been implicated in several diseases including cancer progression, invasion, and metastasis as well as virus survival. There is now a body of evidence that suggests targeting of G3BPs could be explored as a form of cancer therapeutic. This review discusses the important discoveries and advancements made in the field of G3BPs biology over the last two decades including their roles in RNA stability, translational control of cellular transcripts, stress granule formation, cancer progression and its interactions with viruses during infection. An emerging theme for G3BPs is their ability to regulate gene expression in response to environmental stimuli, disease progression and virus infection making it an intriguing target for disease therapies.


Assuntos
DNA Helicases/metabolismo , DNA Helicases/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , RNA Helicases/metabolismo , RNA Helicases/fisiologia , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/fisiologia , Estresse Fisiológico/fisiologia , Animais , Proteínas de Transporte/metabolismo , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/genética , Humanos , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais
18.
Int J Mol Sci ; 20(1)2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30626113

RESUMO

Rice foot rot disease caused by the pathogen Dickeya zeae (formerly known as Erwinia chrysanthemi pv. zeae), is a newly emerging damaging bacterial disease in China and the southeast of Asia, resulting in the loss of yield and grain quality. However, the genetic resistance mechanisms mediated by miRNAs to D. zeae are unclear in rice. In the present study, 652 miRNAs including osa-miR396f predicted to be involved in multiple defense responses to D. zeae were identified with RNA sequencing. A total of 79 differentially expressed miRNAs were detected under the criterion of normalized reads ≥10, including 51 known and 28 novel miRNAs. Degradome sequencing identified 799 targets predicted to be cleaved by 168 identified miRNAs. Among them, 29 differentially expressed miRNA and target pairs including miRNA396f-OsGRFs were identified by co-expression analysis. Overexpression of the osa-miR396f precursor in a susceptible rice variety showed enhanced resistance to D. zeae, coupled with significant accumulation of transcripts of osa-miR396f and reduction of its target the Growth-Regulating Factors (OsGRFs). Taken together, these findings suggest that miRNA and targets including miR396f-OsGRFs have a role in resisting the infections by bacteria D. zeae.


Assuntos
Resistência à Doença , MicroRNAs/metabolismo , Oryza/genética , Oryza/microbiologia , Pectobacterium/fisiologia , Estabilidade de RNA , RNA de Plantas/metabolismo , Análise de Sequência de RNA , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Ontologia Genética , MicroRNAs/química , MicroRNAs/genética , Conformação de Ácido Nucleico , Estabilidade de RNA/genética , RNA de Plantas/química , RNA de Plantas/genética , Reprodutibilidade dos Testes
19.
Int J Oncol ; 54(3): 955-965, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30664186

RESUMO

RNA­binding proteins (RBPs) play a fundamental role in the recurrence and metastasis of colorectal cancer (CRC). In this study, we identified muscleblind­like 1 (MBNL1), an RBP implicated in developmental control, as a robust suppressor of CRC cell metastasis in vitro. By using a scratch assay coupled with time­lapse live cell imaging, our findings revealed that the knockdown of MBNL1 induced epithelial­to­mesenchymal transition (EMT)­like morphological changes in the HCT­116 cells, accompanied by an enhanced cell motility, and by the downregulation of E­cadherin and the upregulation of Snail expression. By contrast, the ectopic overexpression of MBNL1 suppressed EMT, characterized by the upregulation of E­cadherin and the downregulation of Snail expression. Mechanistically, Snail rather than E­cadherin, was identified as a direct downstream target gene of MBNL1. The ectopic the overexpression of MBNL1 markedly enhanced the recruitment of Snail transcripts to processing bodies (P­bodies), leading to the increased degradation of Snail mRNA and consequent translational silencing. Furthermore, the effect of MBNL1 on CRC cell migration was confirmed in additional CRC cell lines. SW480 and HT­29 cells exhibited similar changes in migratory capacity and the expression of Snail/E­cadherin to those observed in HCT­116 cells. On the whole, this study demonstrates that MBNL1 destabilizes Snail transcripts and, in turn, suppresses the EMT of CRC cells through the Snail/E­cadherin axis in vitro. Therefore, this EMT­related MBNL1/Snail/E­cadherin axis may prove to be a novel therapeutic target for CRC metastasis.


Assuntos
Antígenos CD/genética , Caderinas/genética , Neoplasias Colorretais/patologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição da Família Snail/genética , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética
20.
Genes Dev ; 33(3-4): 236-252, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30692204

RESUMO

The multisubunit CCR4-NOT mRNA deadenylase complex plays important roles in the posttranscriptional regulation of gene expression. The NOT4 E3 ubiquitin ligase is a stable component of the CCR4-NOT complex in yeast but does not copurify with the human or Drosophila melanogaster complex. Here we show that the C-terminal regions of human and D. melanogaster NOT4 contain a conserved sequence motif that directly binds the CAF40 subunit of the CCR4-NOT complex (CAF40-binding motif [CBM]). In addition, nonconserved sequences flanking the CBM also contact other subunits of the complex. Crystal structures of the CBM-CAF40 complex reveal a mutually exclusive binding surface for NOT4 and Roquin or Bag of marbles mRNA regulatory proteins. Furthermore, CAF40 depletion or structure-guided mutagenesis to disrupt the NOT4-CAF40 interaction impairs the ability of NOT4 to elicit decay of tethered reporter mRNAs in cells. Together with additional sequence analyses, our results reveal the molecular basis for the association of metazoan NOT4 with the CCR4-NOT complex and show that it deviates substantially from yeast. They mark the NOT4 ubiquitin ligase as an ancient but nonconstitutive cofactor of the CCR4-NOT deadenylase with potential recruitment and/or effector functions.


Assuntos
Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas/fisiologia , Receptores CCR4/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Sequência Conservada , Cristalização , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Estrutura Quaternária de Proteína , Estabilidade de RNA/genética , Receptores CCR4/química , Fatores de Transcrição/genética
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