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1.
Antioxidants (Basel) ; 13(6)2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38929094

RESUMO

Pseudomonas aeruginosa (PA) is an opportunistic pathogen frequently isolated from cutaneous chronic wounds. How PA, in the presence of oxidative stress (OS), colonizes chronic wounds and forms a biofilm is still unknown. The purpose of this study is to investigate the changes in gene expression seen when PA is challenged with the high levels of OS present in chronic wounds. We used a biofilm-forming PA strain isolated from the chronic wounds of our murine model (RPA) and performed a qPCR to obtain gene expression patterns as RPA developed a biofilm in vitro in the presence of high levels of OS, and then compared the findings in vivo, in our mouse model of chronic wounds. We found that the planktonic bacteria under OS conditions overexpressed quorum sensing genes that are important for the bacteria to communicate with each other, antioxidant stress genes important to reduce OS in the microenvironment for survival, biofilm formation genes and virulence genes. Additionally, we performed RNAseq in vivo and identified the activation of novel genes/pathways of the Type VI Secretion System (T6SS) involved in RPA pathogenicity. In conclusion, RPA appears to survive the high OS microenvironment in chronic wounds and colonizes these wounds by turning on virulence, biofilm-forming and survival genes. These findings reveal pathways that may be promising targets for new therapies aimed at disrupting PA-containing biofilms immediately after debridement to facilitate the treatment of chronic human wounds.

3.
Nature ; 623(7987): 580-587, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37938769

RESUMO

Microsatellite repeat expansions within genes contribute to a number of neurological diseases1,2. The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology3-9. Here we reveal that the adenosine in CAG repeat RNA can be methylated to N1-methyladenosine (m1A) by TRMT61A, and that m1A can be demethylated by ALKBH3. We also observed that the m1A/adenosine ratio in CAG repeat RNA increases with repeat length, which is attributed to diminished expression of ALKBH3 elicited by the repeat RNA. Additionally, TDP-43 binds directly and strongly with m1A in RNA, which stimulates the cytoplasmic mis-localization and formation of gel-like aggregates of TDP-43, resembling the observations made for the protein in neurological diseases. Moreover, m1A in CAG repeat RNA contributes to CAG repeat expansion-induced neurodegeneration in Caenorhabditis elegans and Drosophila. In sum, our study offers a new paradigm of the mechanism through which nucleotide repeat expansion contributes to neurological diseases and reveals a novel pathological function of m1A in RNA. These findings may provide an important mechanistic basis for therapeutic intervention in neurodegenerative diseases emanating from CAG repeat expansion.


Assuntos
Adenosina , Caenorhabditis elegans , Proteínas de Ligação a DNA , Drosophila melanogaster , Doenças Neurodegenerativas , RNA , Expansão das Repetições de Trinucleotídeos , Animais , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , RNA/química , RNA/genética , RNA/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , Citoplasma/metabolismo , Modelos Animais de Doenças
4.
Front Immunol ; 14: 1150971, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37090722

RESUMO

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are extremely promising nanoscale cell-free therapeutic agents. We previously identified that intravenous administration (IV) of human umbilical cord MSC-EVs (hUCMSC-EVs), especially hypoxic hUCMSC-EVs (Hypo-EVs), could suppress allergic airway inflammation and remodeling. Here, we further investigated the therapeutic effects of Hypo-EVs administration by atomizing inhalation (INH), which is a non-invasive and efficient drug delivery method for lung diseases. We found that nebulized Hypo-EVs produced by the atomization system (medical/household air compressor and nebulizer) maintained excellent structural integrity. Nebulized Dir-labeled Hypo-EVs inhaled by mice were mainly restricted to lungs. INH administration of Hypo-EVs significantly reduced the airway inflammatory infiltration, decreased the levels of IL-4, IL-5 and IL-13 in bronchoalveolar lavage fluid (BALF), declined the content of OVA-specific IgE in serum, attenuated the goblet cell metaplasia, and the expressions of subepithelial collagen-1 and α-smooth muscle actin (α-SMA). Notably, Hypo-EV INH administration was generally more potent than Hypo-EV IV in suppressing IL-13 levels and collagen-1 and α-SMA expressions. RNA sequencing revealed that various biological processes, such as cell adhesion, innate immune response, B cell activation, and extracellular space, were associated with the activity of Hypo-EV INH against asthma mice. In addition, Hypo-EVs could load exogenous miR-146a-5p (miR-146a-5p-EVs). Furthermore, INH administration of miR-146a-5p-EVs resulted in a significantly increased expression of miR-146a-5p mostly in lungs, and offered greater protection against the OVA-induced increase in airway inflammation, subepithelial collagen accumulation and myofibroblast compared with nebulized Hypo-EVs. Overall, nebulized Hypo-EVs effectively attenuated allergic airway inflammation and remodeling, potentially creating a non-invasive route for the use of MSC-EVs in asthma treatment.


Assuntos
Asma , Vesículas Extracelulares , MicroRNAs , Humanos , Animais , Camundongos , Interleucina-13 , Inflamação/terapia , Vesículas Extracelulares/metabolismo , Colágeno Tipo I , Colágeno/metabolismo , Hipóxia , MicroRNAs/genética , MicroRNAs/uso terapêutico
5.
Nat Commun ; 12(1): 5948, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34642330

RESUMO

Skeletal muscle is a highly adaptable tissue and remodels in response to exercise training. Using short RNA sequencing, we determine the miRNA profile of skeletal muscle from healthy male volunteers before and after a 14-day aerobic exercise training regime. Among the exercise training-responsive miRNAs identified, miR-19b-3p was selected for further validation. Overexpression of miR-19b-3p in human skeletal muscle cells increases insulin signaling, glucose uptake, and maximal oxygen consumption, recapitulating the adaptive response to aerobic exercise training. Overexpression of miR-19b-3p in mouse flexor digitorum brevis muscle enhances contraction-induced glucose uptake, indicating that miR-19b-3p exerts control on exercise training-induced adaptations in skeletal muscle. Potential targets of miR-19b-3p that are reduced after aerobic exercise training include KIF13A, MAPK6, RNF11, and VPS37A. Amongst these, RNF11 silencing potentiates glucose uptake in human skeletal muscle cells. Collectively, we identify miR-19b-3p as an aerobic exercise training-induced miRNA that regulates skeletal muscle glucose metabolism.


Assuntos
Proteínas de Ligação a DNA/genética , Exercício Físico/fisiologia , Glucose/metabolismo , MicroRNAs/genética , Processamento de Proteína Pós-Traducional , Adulto , Animais , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Metabolismo Energético/genética , Voluntários Saudáveis , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Proteína Quinase 6 Ativada por Mitógeno/genética , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Consumo de Oxigênio/genética , Fosforilação , Condicionamento Físico Animal , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
6.
Nat Cell Biol ; 23(4): 424-436, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33820973

RESUMO

Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs-mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)-that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation.


Assuntos
Processamento Pós-Transcricional do RNA/genética , Pequeno RNA não Traduzido/genética , RNA-Seq , Transcriptoma/genética , DNA Complementar/genética , Células HeLa , Humanos , MicroRNAs/genética , RNA Ribossômico/genética
7.
PLoS Genet ; 17(4): e1009511, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33826611

RESUMO

Once loaded onto Argonaute proteins, microRNAs form a silencing complex called miRISC that targets mostly the 3'UTR of mRNAs to silence their translation. How microRNAs are transported to and from their target mRNA remains poorly characterized. While some reports linked intracellular trafficking to microRNA activity, it is still unclear how these pathways coordinate for proper microRNA-mediated gene silencing and turnover. Through a forward genetic screen using Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the microRNA pathway. We show that TBC-11 acts mainly through the small GTPase RAB-6 and that its regulation is required for microRNA function. The absence of functional TBC-11 increases the pool of microRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes. Furthermore, in this condition, this pool of Argonaute accumulates in a perinuclear region and forms a high molecular weight complex. Altogether, our data suggest that the alteration of TBC-11 generates a fraction of ALG-1 that cannot bind to target mRNAs, leading to defective gene repression. Our results establish the importance of intracellular trafficking for microRNA function and demonstrate the involvement of a small GTPase and its GAP in proper Argonaute localization in vivo.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Proteínas rab de Ligação ao GTP/genética , Regiões 3' não Traduzidas/genética , Animais , Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Inativação Gênica , MicroRNAs/genética , RNA Mensageiro/genética
9.
Mol Cell ; 81(3): 546-557.e5, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33378643

RESUMO

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


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Processamento Pós-Transcricional do RNA , RNA/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Monoéster Fosfórico Hidrolases/genética , Fosforilação , RNA/genética , Capuzes de RNA , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Espermatogênese , Especificidade por Substrato
10.
Viruses ; 12(11)2020 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-33171824

RESUMO

Non-coding small RNAs play important roles in virus-host interactions. For hosts, small RNAs can serve as sensors in antiviral pathways including RNAi and CRISPR; for viruses, small RNAs can be involved in viral transcription and replication. This paper covers several recent discoveries on small RNA mediated virus-host interactions, and focuses on influenza virus cap-snatching and a few important virus sensors including PIR-1, RIG-I like protein DRH-1 and piRNAs. The paper also discusses recent advances in mammalian antiviral RNAi.


Assuntos
Interações entre Hospedeiro e Microrganismos/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Viral/genética , Animais , Camundongos , Replicação Viral
11.
Gene Technol ; 9(2)2020 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-32953938

RESUMO

High-throughput sequencing has become a standard and powerful tool for analyzing nucleic acids primarily due to its sensitivity and convenience. Small RNAs play important roles in regulating cellular and viral genes. The conventional methods for small RNA analyses are tedious and often lack accuracy, specificity and sensitivity for many small RNA species. Therefore, high-throughput sequencing becomes an indispensable tool for analyzing small RNAs. However, it is challenging to generate a reliable and representative small RNA library for high-throughput sequencing since small RNAs are usually expressed at extremely low levels and often contain modifications which affect library construction, usually causing biased readouts. This review compares various strategies for generating small RNA libraries of high quality and reliability, and provides recommendations on best practice in preparing high-throughput sequencing RNA libraries.

12.
RNA ; 26(9): 1170-1183, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32444459

RESUMO

Influenza A virus (IAV) utilizes cap-snatching to obtain host capped small RNAs for priming viral mRNA synthesis, generating capped hybrid mRNAs for translation. Previous studies have been focusing on canonical cap-snatching, which occurs at the very 5' end of viral mRNAs. Here we discovered noncanonical cap-snatching, which generates capped hybrid mRNAs/noncoding RNAs mapped to the region ∼300 nucleotides (nt) upstream of each mRNA 3' end, and to the 5' region, primarily starting at the second nt, of each virion RNAs (vRNA). Like canonical cap-snatching, noncanonical cap-snatching utilizes a base-pairing between the last nt G of host capped RNAs and a nt C of template RNAs to prime RNA synthesis. However, the nt upstream of this template C is usually A/U rather than just U; prime-realignment occurs less frequently. We also demonstrate that IAV can snatch capped IAV RNAs in addition to host RNAs. Noncanonical cap-snatching likely generates novel mRNAs with start AUG encoded in viral or host RNAs. These findings expand our understanding of cap-snatching mechanisms and suggest that IAV may utilize noncanonical cap-snatching to diversify its mRNAs/ncRNAs.


Assuntos
Vírus da Influenza A/genética , Capuzes de RNA/genética , RNA Mensageiro/genética , RNA não Traduzido/genética , Células A549 , Pareamento de Bases/genética , Linhagem Celular Tumoral , Humanos , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Transcrição Gênica/genética
13.
RNA ; 26(2): 218-227, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31754076

RESUMO

High-throughput sequencing has become a standard tool for analyzing RNA and DNA. This method usually needs a cDNA/DNA library ligated with specific 5' and 3' linkers. Unlike mRNA, small RNA often contains modifications including 5' cap or triphosphate and 2'-O-methyl, requiring additional processing steps before linker additions during cloning processes; due to low expression levels, it is difficult to clone small RNA with a small amount of total RNA. Here we present a new strategy to clone 5' modified or unmodified small RNA in an all-liquid-based reaction carried out in a single PCR tube with as little as 20 ng total RNA. The 7-h cloning process only needs ∼1 h of labor. Moreover, this method can also clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA; the barcoded PCR primers are also compatible with non-cDNA cloning applications, including the preparation of genomic libraries. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, versatile, and cost-effective. Moreover, the all-liquid-based reaction can be performed in an automated manner.


Assuntos
Caenorhabditis elegans/genética , Clonagem Molecular , Sequenciamento de Nucleotídeos em Larga Escala , RNA de Helmintos/genética , Animais , Primers do DNA/genética , DNA Complementar/genética , Biblioteca Gênica , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Sensibilidade e Especificidade , Análise de Sequência de RNA
14.
Anal Chem ; 92(1): 1346-1354, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31815440

RESUMO

5-Methylcytosine is found in both DNA and RNA; although its functions in DNA are well established, the exact role of 5-methylcytidine (m5C) in RNA remains poorly defined. Here we identified, by employing a quantitative proteomics method, multiple candidate recognition proteins of m5C in RNA, including several YTH domain-containing family (YTHDF) proteins. We showed that YTHDF2 could bind directly to m5C in RNA, albeit at a lower affinity than that toward N6-methyladenosine (m6A) in RNA, and this binding involves Trp432, a conserved residue located in the hydrophobic pocket of YTHDF2 that is also required for m6A recognition. RNA bisulfite sequencing results revealed that, after CRISPR-Cas9-mediated knockout of the YTHDF2 gene, the majority of m5C sites in rRNA (rRNA) exhibited substantially augmented levels of methylation. Moreover, we found that YTHDF2 is involved in pre-rRNA processing in cells. Together, our data expanded the functions of the YTHDF2 protein in post-transcriptional regulations of RNA and provided novel insights into the functions of m5C in RNA biology.


Assuntos
5-Metilcitosina/química , RNA Ribossômico/química , Proteínas de Ligação a RNA/química , 5-Metilcitosina/metabolismo , Sítios de Ligação , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Metilação , Estrutura Molecular , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
15.
Cell Host Microbe ; 25(1): 153-165.e5, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30595554

RESUMO

RNA silencing (RNAi) has a well-established role in anti-viral immunity in plants. The destructive eukaryotic pathogen Phytophthora encodes suppressors of RNAi (PSRs), which enhance plant susceptibility. However, the role of small RNAs in defense against eukaryotic pathogens is unclear. Here, we show that Phytophthora infection of Arabidopsis leads to increased production of a diverse pool of secondary small interfering RNAs (siRNAs). Instead of regulating endogenous plant genes, these siRNAs are found in extracellular vesicles and likely silence target genes in Phytophthora during natural infection. Introduction of a plant siRNA in Phytophthora leads to developmental deficiency and abolishes virulence, while Arabidopsis mutants defective in secondary siRNA biogenesis are hypersusceptible. Notably, Phytophthora effector PSR2 specifically inhibits secondary siRNA biogenesis in Arabidopsis and promotes infection. These findings uncover the role of siRNAs as antimicrobial agents against eukaryotic pathogens and highlight a defense/counter-defense arms race centered on trans-kingdom gene silencing between hosts and pathogens.


Assuntos
Arabidopsis/imunologia , Suscetibilidade a Doenças/microbiologia , Phytophthora/metabolismo , Phytophthora/patogenicidade , Doenças das Plantas/imunologia , Interferência de RNA/imunologia , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes Reporter/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , MicroRNAs/genética , MicroRNAs/imunologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Imunidade Vegetal/imunologia , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/efeitos dos fármacos , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Nicotiana , Verticillium , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
17.
Curr Biol ; 27(6): 795-806, 2017 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-28262484

RESUMO

The recent discovery of the positive-sense single-stranded RNA (ssRNA) Orsay virus (OV) as a natural pathogen of the nematode Caenorhabditis elegans has stimulated interest in exploring virus-nematode interactions. However, OV infection is restricted to a small number of intestinal cells, even in nematodes defective in their antiviral RNA interference (RNAi) response, and is neither lethal nor vertically transmitted. Using a fluorescent reporter strain of the negative-sense ssRNA vesicular stomatitis virus (VSV), we show that microinjection of VSV particles leads to a dose-dependent, muscle tissue-tropic, lethal infection in C. elegans. Furthermore, we find nematodes deficient for components of the antiviral RNAi pathway, such as Dicer-related helicase 1 (DRH-1), to display hypersusceptibility to VSV infection as evidenced by elevated infection rates, virus replication in multiple tissue types, and earlier mortality. Strikingly, infection of oocytes and embryos could also be observed in drh-1 mutants. Our results suggest that the antiviral RNAi response not only inhibits vertical VSV transmission but also promotes transgenerational inheritance of antiviral immunity. Our study introduces a new, in vivo virus-host model system for exploring arbovirus pathogenesis and provides the first evidence for vertical pathogen transmission in C. elegans.


Assuntos
Infecções por Arbovirus/transmissão , Transmissão Vertical de Doenças Infecciosas , Interferência de RNA , Infecções por Rhabdoviridae/transmissão , Vesiculovirus/fisiologia , Animais , Infecções por Arbovirus/virologia , Caenorhabditis elegans , Microinjeções , Infecções por Rhabdoviridae/virologia
18.
RNA ; 22(10): 1492-9, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27495319

RESUMO

Gld2, a noncanonical cytoplasmic poly(A) polymerase, interacts with the RNA binding protein CPEB1 to mediate polyadenylation-induced translation in dendrites of cultured hippocampal neurons. Depletion of Gld2 from the hippocampus leads to a deficit in long-term potentiation evoked by theta burst stimulation. At least in mouse liver and human primary fibroblasts, Gld2 also 3' monoadenylates and thereby stabilizes specific miRNAs, which enhance mRNA translational silencing and eventual destruction. These results suggest that Gld2 would be likely to monoadenylate and stabilize miRNAs in the hippocampus, which would produce measurable changes in animal behavior. We now report that using Gld2 knockout mice, there are detectable alterations in specific miRNA monoadenylation in the hippocampus when compared to wild type, but that these modifications produce no detectable effect on miRNA stability. Moreover, we surprisingly find no overt change in animal behavior when comparing Gld2 knockout to wild-type mice. These data indicate that miRNA monoadenylation-mediated stability is cell type-specific and that monoadenylation has no measurable effect on higher cognitive function.


Assuntos
Comportamento Animal , Hipocampo/metabolismo , MicroRNAs/genética , Polinucleotídeo Adenililtransferase/metabolismo , Processamento de Terminações 3' de RNA , Animais , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Polinucleotídeo Adenililtransferase/genética , Estabilidade de RNA
19.
RNA ; 21(12): 2067-75, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26428694

RESUMO

Influenza A virus (IAV) lacks the enzyme for adding 5' caps to its RNAs and snatches the 5' ends of host capped RNAs to prime transcription. Neither the preference of the host RNA sequences snatched nor the effect of cap-snatching on host processes is completely defined. Previous studies of influenza cap-snatching used poly(A)-selected RNAs from infected cells or relied on annotated host genes to define the snatched host RNAs, and thus lack details on many noncoding host RNAs including snRNAs, snoRNAs, and promoter-associated capped small (cs)RNAs, which are made by "paused" Pol II during transcription initiation. In this study, we used a nonbiased technique, CapSeq, to identify host and viral-capped RNAs including nonpolyadenylated RNAs in the same samples, and investigated the substrate-product correlation between the host RNAs and the viral RNAs. We demonstrated that noncoding host RNAs, particularly U1 and U2, are the preferred cap-snatching source over mRNAs or pre-mRNAs. We also found that csRNAs are highly snatched by IAV. Because the functions of csRNAs remain mostly unknown, especially in somatic cells, our finding reveals that csRNAs at least play roles in the process of IAV infection. Our findings support a model where nascent RNAs including csRNAs are the preferred targets for cap-snatching by IAV and raise questions about how IAV might use snatching preferences to modulate host-mRNA splicing and transcription.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Capuzes de RNA/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica , Genes Virais , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo
20.
Genes Dev ; 29(4): 362-78, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25691467

RESUMO

Approximately 75% of the human genome is transcribed, the majority of which does not encode protein. However, many noncoding RNAs (ncRNAs) are rapidly degraded after transcription, and relatively few have established functions, questioning the significance of this observation. Here we show that esBAF, a SWI/SNF family nucleosome remodeling factor, suppresses transcription of ncRNAs from ∼57,000 nucleosome-depleted regions (NDRs) throughout the genome of mouse embryonic stem cells (ESCs). We show that esBAF functions to both keep NDRs nucleosome-free and promote elevated nucleosome occupancy adjacent to NDRs. Reduction of adjacent nucleosome occupancy upon esBAF depletion is strongly correlated with ncRNA expression, suggesting that flanking nucleosomes form a barrier to pervasive transcription. Upon forcing nucleosome occupancy near two NDRs using a nucleosome-positioning sequence, we found that esBAF is no longer required to silence transcription. Therefore, esBAF's function to enforce nucleosome occupancy adjacent to NDRs, and not its function to maintain NDRs in a nucleosome-free state, is necessary for silencing transcription over ncDNA. Finally, we show that the ability of a strongly positioned nucleosome to repress ncRNA depends on its translational positioning. These data reveal a novel role for esBAF in suppressing pervasive transcription from open chromatin regions in ESCs.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/fisiologia , RNA não Traduzido/genética , Animais , Montagem e Desmontagem da Cromatina , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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