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1.
Mol Biol (Mosk) ; 53(3): 476-484, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184613

RESUMO

It is known that long (200-300 nucleotides and longer) non-protein-coding RNAs (ncRNAs) tissue-specifically expressed from the regulatory regions of developmental genes can regulate the transcription of the mRNA of these genes. In this study, an attempt is made to identify differentially expressed ncRNAs in the extended promoter region of the fork head (fkh) gene of the fruit fly Drosophila melanogaster. We investigated four preparations of the total RNA: from embryos, from adult flies (separately from females and males), and from the S2 cell line of cultured Drosophila cells. In the total RNA preparations from embryos and adult flies, the levels of fkh expression differed substantially, whereas in S2 cells its expression is not detected at all (shown in this work). We perform classical Northern blot analysis of gel-separated RNAs hybridized to a series of radioactively labeled DNA fragments corresponding to the adjacent and partially overlapping regions of the promoter region of the fkh gene. Several previously unknown differentially expressed ncRNAs are detected, including those in the regions overlapping with the previously detected regulatory elements (TRE1 and salivary gland enhancer sgE) and the transcription start site of the fkh gene. The collected data complement and clarify the results of the previously conducted RNA-seq experiments, in particular, in terms of the length of the detected RNAs. These results may serve as a foundation for further studies of the mechanisms of tissue-specific regulation of the fkh gene expression.


Assuntos
Northern Blotting , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante/análise , RNA Longo não Codificante/genética , Animais , Linhagem Celular , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Elementos Facilitadores Genéticos/genética , Feminino , Masculino , Especificidade de Órgãos
2.
Nucleic Acids Res ; 47(14): 7321-7332, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31214713

RESUMO

AntimiR is an antisense oligonucleotide that has been developed to silence microRNA (miRNA) for the treatment of intractable diseases. Enhancement of its in vivo efficacy and improvement of its toxicity are highly desirable but remain challenging. We here design heteroduplex oligonucleotide (HDO)-antimiR as a new technology comprising an antimiR and its complementary RNA. HDO-antimiR binds targeted miRNA in vivo more efficiently by 12-fold than the parent single-stranded antimiR. HDO-antimiR also produced enhanced phenotypic effects in mice with upregulated expression of miRNA-targeting messenger RNAs. In addition, we demonstrated that the enhanced potency of HDO-antimiR was not explained by its bio-stability or delivery to the targeted cell, but reflected an improved intracellular potency. Our findings provide new insights into biology of miRNA silencing by double-stranded oligonucleotides and support the in vivo potential of this technology based on a new class of for the treatment of miRNA-related diseases.


Assuntos
DNA de Cadeia Simples/genética , Inativação Gênica , MicroRNAs/genética , Ácidos Nucleicos Heteroduplexes/genética , Oligonucleotídeos Antissenso/genética , Animais , Northern Blotting , DNA de Cadeia Simples/metabolismo , Feminino , Regulação da Expressão Gênica , Rim/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos ICR , MicroRNAs/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , Ácidos Nucleicos Heteroduplexes/farmacocinética , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacocinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Baço/metabolismo
3.
Chin Med J (Engl) ; 132(13): 1591-1598, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31205077

RESUMO

BACKGROUND: Natural anti-sense transcripts (NATs), which are transcribed from the complementary DNA strand of annotated genes, exert regulatory function of gene expression. Increasing studies recognized anti-sense transcription widespread throughout human cytomegalovirus (HCMV) genome, whereas the anti-sense transcription of RNA1.2 gene locus has never been investigated. In this study, the transcription of the RNA1.2 anti-sense strand was investigated in clinically isolated HCMV strain. METHODS: Strand-specific high-through RNA-sequencing (RNA-seq) was performed to find possible anti-sense transcripts (ASTs). For analyzing and visualization of RNA-seq data sets, Integrative Genomics Viewer software was applied. To confirm these possibilities, Northern blotting and rapid amplification of cDNA ends (RACE) were used. RESULTS: Transcription of the opposite strand of RNA1.2 gene locus was detected by RNA-sequencing using RNAs extracted from human embryonic lung fibroblasts infected with HCMV clinical isolate HAN. At least three HCMV NATs, named RNA1.2 AST 1, RNA1.2 AST2, and RNA1.2 AST3, were characterized by Northern blotting and RACE analyses. These RNA1.2 ASTs orientated from the complementary strand of RNA1.2 locus during the late phase of HCMV infection. The 5'- and 3'-termini of these transcripts were located within the opposite sequence of the predicted RNA1.2 gene. CONCLUSION: A cluster of novel NATs was transcribed from the opposite sequence of the HCMV RNA1.2 gene region.


Assuntos
Citomegalovirus/genética , RNA Antissenso/genética , Northern Blotting , Células Cultivadas , Genoma Viral/genética , Humanos , Software
4.
Nucleic Acids Res ; 47(11): 5936-5949, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30997502

RESUMO

In eukaryotes and archaea, tRNA genes frequently contain introns, which are removed during maturation. However, biological roles of tRNA introns remain elusive. Here, we constructed a complete set of Saccharomyces cerevisiae strains in which the introns were removed from all the synonymous genes encoding 10 different tRNA species. All the intronless strains were viable, but the tRNAPheGAA and tRNATyrGUA intronless strains displayed slow growth, cold sensitivity and defective growth under respiratory conditions, indicating physiological importance of certain tRNA introns. Northern analyses revealed that removal of the introns from genes encoding three tRNAs reduced the amounts of the corresponding mature tRNAs, while it did not affect aminoacylation. Unexpectedly, the tRNALeuCAA intronless strain showed reduced 5.8S rRNA levels and abnormal nucleolar morphology. Because pseudouridine (Ψ) occurs at position 34 of the tRNAIleUAU anticodon in an intron-dependent manner, tRNAIleUAU in the intronless strain lost Ψ34. However, in a portion of tRNAIleUAU population, position 34 was converted into 5-carbamoylmethyluridine (ncm5U), which could reduce decoding fidelity. In summary, our results demonstrate that, while introns are dispensable for cell viability, some introns have diverse roles, such as ensuring proper growth under various conditions and controlling the appropriate anticodon modifications for accurate pairing with the codon.


Assuntos
Íntrons , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticódon , Northern Blotting , Códon , Genoma Fúngico , Leucina/química , Mutação , Conformação de Ácido Nucleico , Fenótipo , Plasmídeos/metabolismo , Pseudouridina , RNA/química , Processamento Pós-Transcricional do RNA , RNA Fúngico/metabolismo , RNA Ribossômico 5,8S/metabolismo
5.
Int J Mol Med ; 43(6): 2267-2278, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31017262

RESUMO

Among a number of mRNA modifications, N6­methyladenosine (m6A) modification is the most common type in eukaryotes and nuclear­replicating viruses. m6A has a significant role in numerous cancer types, including leukemia, brain tumors, liver cancer, breast cancer and lung cancer. Although m6A methyltransferases are essential during RNA modifications, the biological functions of m6A and the underlying mechanisms remain to be fully elucidated, predominantly due to the limited detection methods for m6A. In the present review, the currently available m6A detection methods and the respective scope of their applications are presented to facilitate the further investigation of the roles of m6A in biological process.


Assuntos
Adenosina/análogos & derivados , RNA/química , Adenosina/análise , Adenosina/genética , Animais , Técnicas Biossensoriais/métodos , Northern Blotting/métodos , Cromatografia Líquida de Alta Pressão/métodos , Técnicas Eletroquímicas/métodos , Humanos , Immunoblotting/métodos , Imunoprecipitação/métodos , Metilação , Neoplasias/genética , RNA/genética , Análise de Sequência de RNA/métodos
6.
Nucleic Acids Res ; 47(7): 3353-3364, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30820533

RESUMO

While the number of human miRNA candidates continuously increases, only a few of them are completely characterized and experimentally validated. Toward determining the total number of true miRNAs, we employed a combined in silico high- and experimental low-throughput validation strategy. We collected 28 866 human small RNA sequencing data sets containing 363.7 billion sequencing reads and excluded falsely annotated and low quality data. Our high-throughput analysis identified 65% of 24 127 mature miRNA candidates as likely false-positives. Using northern blotting, we experimentally validated miRBase entries and novel miRNA candidates. By exogenous overexpression of 108 precursors that encode 205 mature miRNAs, we confirmed 68.5% of the miRBase entries with the confirmation rate going up to 94.4% for the high-confidence entries and 18.3% of the novel miRNA candidates. Analyzing endogenous miRNAs, we verified the expression of 8 miRNAs in 12 different human cell lines. In total, we extrapolated 2300 true human mature miRNAs, 1115 of which are currently annotated in miRBase V22. The experimentally validated miRNAs will contribute to revising targetomes hypothesized by utilizing falsely annotated miRNAs.


Assuntos
Simulação por Computador , MicroRNAs/análise , MicroRNAs/genética , Análise de Sequência de RNA , Northern Blotting , Linhagem Celular , Conjuntos de Dados como Assunto , Reações Falso-Positivas , Humanos , MicroRNAs/isolamento & purificação , Anotação de Sequência Molecular , Precursores de RNA/análise , Precursores de RNA/genética , Reprodutibilidade dos Testes
7.
Plant Physiol Biochem ; 137: 62-74, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30738218

RESUMO

Shortfall of rain that creates drought like situation in non-irrigated agriculture system often limits rice production, necessitating introduction of drought tolerance trait into the cultivar of interest. The mechanism governing drought tolerance is, however, largely unknown, particularly the involvement of miRNAs, the master regulators of biochemical events. In this regard, response study on a drought tolerant rice variety KMJ 1-12-3 to 20% PEG (osmolality- 315 mOsm/kg) as drought stress revealed significant changes in abundance of several conserved miRNAs targeting transcription factors like homeodomain-leucine zipper, MADS box family protein, C2H2 zinc finger protein and Myb, well known for their importance in drought tolerance in plants. The response study also revealed significant PEG-induced decrease in abundance of the miRNAs targeting cyclin A, cyclin-dependent kinase, guanine nucleotide exchange factor, GTPase-activating protein, 1-aminocyclopropane-1-carboxylic acid oxidase and indole-3-acetic beta-glucosyl transferase indicating miRNA-regulated role of the cell cycle regulators, G-protein signalling and the plant hormones ethylene and IAA in drought tolerance in plants. The study confirmed the existence of four novel miRNAs, including osa-miR12470, osa-miR12471, osa-miR12472 and osa-miR12473, and the targets of three of them could be successfully validated. The PEG-induced decrease in abundance of the novel miRNAs osa-miR12470 and osa-miR12473 targeting RNA dependent RNA polymerase and equilibrative nucleoside transporter, respectively suggested an overall increase in both degradation and synthesis of nucleic acids in plants challenged with drought stress. The drought-responsive miRNAs identified in the study may be proved useful in introducing the trait in the rice cultivars of choice by manipulation of their cellular abundance.


Assuntos
Secas , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Oryza/fisiologia , Proteínas de Plantas/genética , Northern Blotting , Etilenos/metabolismo , Oryza/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética
8.
mBio ; 10(1)2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755517

RESUMO

Herpes simplex virus 1 (HSV-1) switches between two infection programs, productive ("lytic") and latent infection. Some HSV-1 microRNAs (miRNAs) have been hypothesized to help control this switch, and yet little is known about regulation of their expression. Using Northern blot analyses, we found that, despite inherent differences in biogenesis efficiency among six HSV-1 miRNAs, all six exhibited high pre-miRNA/miRNA ratios during lytic infection of different cell lines and, when detectable, in acutely infected mouse trigeminal ganglia. In contrast, considerably lower ratios were observed in latently infected ganglia and in cells transduced with lentiviral vectors expressing the miRNAs, suggesting that HSV-1 lytic infection blocks miRNA biogenesis. This phenomenon is not specific to viral miRNAs, as a host miRNA expressed from recombinant HSV-1 also exhibited high pre-miRNA/miRNA ratios late during lytic infection. The levels of most of the mature miRNAs remained stable during infection in the presence of actinomycin D, indicating that the high ratios are due to inefficient pre-miRNA conversion to miRNA. Cellular fractionation experiments showed that late (but not early) during infection, pre-miRNAs were enriched in the nucleus and depleted in the cytoplasm, indicating that nuclear export was blocked. A mutation eliminating ICP27 expression or addition of acyclovir reduced pre-miRNA/miRNA ratios, but mutations drastically reducing Us11 expression did not. Thus, HSV-1 lytic infection inhibits miRNA biogenesis at the step of nuclear export and does so in an ICP27- and viral DNA synthesis-dependent manner. This mechanism may benefit the virus by reducing expression of repressive miRNAs during lytic infection while permitting elevated expression during latency.IMPORTANCE Various mechanisms have been identified by which viruses target host small RNA biogenesis pathways to achieve optimal infection outcomes. Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen whose successful persistence in the host entails both productive ("lytic") and latent infection. Although many HSV-1 miRNAs have been discovered and some are thought to help control the lytic/latent switch, little is known about regulation of their biogenesis. By characterizing expression of both pre-miRNAs and mature miRNAs under various conditions, this study revealed striking differences in miRNA biogenesis between lytic and latent infection and uncovered a regulatory mechanism that blocks pre-miRNA nuclear export and is dependent on viral protein ICP27 and viral DNA synthesis. This mechanism represents a new virus-host interaction that could limit the repressive effects of HSV-1 miRNAs hypothesized to promote latency and may shed light on the regulation of miRNA nuclear export, which has been relatively unexplored.


Assuntos
Transporte Ativo do Núcleo Celular , Herpes Simples/virologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Animais , Northern Blotting , Linhagem Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Herpes Simples/patologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Mutação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Gânglio Trigeminal/patologia , Gânglio Trigeminal/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Latência Viral
9.
Methods Mol Biol ; 1932: 109-120, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30701495

RESUMO

Small RNAs (sRNAs) are RNAs of low abundance in organisms. Among sRNAs, miRNAs are included and represent approximately 10% of the total number of sRNAs. The isolation of sRNAs is critical for miRNA detection and analysis. The precipitation of low-molecular-weight (LMW) RNAs from total RNA extracts has allowed enrichment of sRNAs. Here, we describe a simple method to isolate sRNAs from different plant species. The main advantage of this method is that it does not need first an extraction of total RNA and it is not based on TRIzol® reagent. This method has been successfully used for miRNA analyses by Northern blot assay and RT-qPCR (these techniques are as well described in this chapter), as well as sRNA library preparation.


Assuntos
MicroRNAs/genética , Plantas/genética , RNA de Plantas/genética , Northern Blotting/métodos , Regulação da Expressão Gênica de Plantas/genética , Biblioteca Gênica , RNA Interferente Pequeno/genética
10.
Methods Mol Biol ; 1932: 121-129, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30701496

RESUMO

The study of regulatory small RNAs, such as siRNAs and microRNAs in plants, has necessitated methods tailored to their unique features. Their analysis demands the use of sensitive and quantitative methods for their detection. The use of Northern blot hybridization offers an attractive alternative to address qualitative as well as quantitative features. We highlight the advantages and shortcomings of this method and offer a detailed description of the techniques that best work in our hands, considering their use for the study of several small RNAs in multiple samples. We enumerate relevant details as well as cautionary comments in cases where we have detected potential difficulties.


Assuntos
MicroRNAs/genética , Plantas/genética , RNA de Plantas/genética , Northern Blotting/métodos , Regulação da Expressão Gênica de Plantas/genética , Hibridização de Ácido Nucleico/genética , RNA Interferente Pequeno/genética
11.
Mol Ther ; 27(2): 380-393, 2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30528088

RESUMO

The role of long non-coding RNA (lncRNA) in idiopathic pulmonary fibrosis (IPF) is poorly understood. We found a novel lncRNA-ITPF that was upregulated in IPF. Bioinformatics and in vitro translation verified that lncITPF is an actual lncRNA, and its conservation is in evolution. Northern blot and rapid amplification of complementary DNA ends were used to analyze the full-length sequence of lncITPF. RNA fluorescence in situ hybridization and nucleocytoplasmic separation demonstrated that lncITPF was mainly located in the nucleus. RNA sequencing, chromatin immunoprecipitation (ChIP)-qPCR, CRISPR-Cas9 technology, and promoter activity analysis showed that the fibrotic function of lncITPF depends on its host gene integrin ß-like 1 (ITGBL1), but they did not share the same promoter and were not co-transcribed. Luciferase activity, pathway inhibitors, and ChIP-qPCR showed that smad2/3 binds to the lncITPF promoter, and TGF-ß1-smad2/3 was the upstream inducer of the fibrotic pathway. Furthermore, RNA-protein pull-down, liquid chromatography-mass spectrometry (LC-MS), and protein-RNA immunoprecipitation showed that lncITPF regulated H3 and H4 histone acetylation in the ITGBL1 promoter by targeting heterogeneous nuclear ribonucleoprotein L. Finally, sh-lncITPF was used to evaluate the therapeutic effect of lncITPF. Clinical analysis showed that lncITPF is associated with the clinicopathological features of IPF patients. Our findings provide a therapeutic target or diagnostic biomarker for IPF.


Assuntos
Sistemas CRISPR-Cas/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , RNA Longo não Codificante/metabolismo , Idoso , Animais , Northern Blotting , Western Blotting , Sistemas CRISPR-Cas/genética , Linhagem Celular , Movimento Celular/genética , Movimento Celular/fisiologia , Imunoprecipitação da Cromatina , Cromatografia Líquida , Feminino , Imunofluorescência , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/genética , Humanos , Fibrose Pulmonar Idiopática/genética , Imunoprecipitação , Hibridização In Situ , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Miofibroblastos/citologia , Miofibroblastos/metabolismo , RNA Longo não Codificante/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real
12.
Methods ; 155: 3-9, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30419334

RESUMO

The function and fate of cellular RNAs are often governed by the phosphorylation state at the 5' end or the identity of whatever cap may be present there. Here we describe methods for examining these important 5'-terminal features on any cellular or synthetic RNA of interest that can be detected by Northern blotting. One such method, PABLO, is a splinted ligation assay that makes it possible to accurately quantify the percentage of 5' ends that are monophosphorylated. Another, PACO, is a capping assay that reveals the percentage of 5' ends that are diphosphorylated. A third, boronate gel electrophoresis in conjunction with deoxyribozyme-mediated cleavage, enables different types of caps (e.g., m7Gppp caps versus NAD caps) to be distinguished from one another and the percentage of each to be determined. After completing all three tests, the percentage of 5' ends that are triphosphorylated can be deduced by process of elimination. Together, this battery of assays allows the 5' terminus of an RNA to be profiled in unprecedented detail.


Assuntos
Região 5'-Flanqueadora , Eletroforese em Gel de Poliacrilamida/métodos , Capuzes de RNA/análise , Edição de RNA , RNA Mensageiro/química , Northern Blotting/métodos , Ácidos Borônicos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfatos/metabolismo , Fosforilação , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
13.
Planta ; 249(2): 457-468, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30251012

RESUMO

MAIN CONCLUSION: In this study, we show that aberrant pre-mRNAs from non-spliced and non-polyadenylated intron-containing transgenes are channelled to the RNA silencing pathway. In plants, improperly processed transcripts are called aberrant RNAs (ab-RNAs) and are eliminated by either RNA silencing or RNA decay mechanisms. Ab-RNAs transcribed from intronless genes are copied by RNA-directed RNA polymerases (RDRs) into double-stranded RNAs which are subsequently cleaved by DICER-LIKE endonucleases into small RNAs (sRNAs). In contrast, ab-RNAs from intron-containing genes are suggested to be channelled post-splicing to exonucleolytic degradation. Yet, it is not clear how non-spliced aberrant pre-mRNAs are eliminated. We reasoned that transient expression of agroinfiltrated intron-containing transgenes in Nicotiana benthamiana would allow us to study the steady-state levels of non-spliced pre-mRNAs. SRNA deep sequencing of the agroinfiltrated transgenes revealed the presence of sRNAs mapping to the entire non-spliced pre-mRNA suggesting that RDRs (most likely RDR6) processed aberrant non-spliced pre-mRNAs. Primary and secondary sRNAs with lengths of 18-25 nucleotides (nt) were detected, with the most prominent sRNA size class of 22 nt. SRNAs also mapped to the terminator sequence, indicating that RDR substrates also comprised read-through transcripts devoid of polyadenylation tail. Importantly, the occurring sRNAs efficiently targeted cognate mRNA for degradation but failed to cleave the non-spliced pre-mRNA, corroborating the notion that sRNAs are not triggering RNA cleavage in the nucleus.


Assuntos
Íntrons , Precursores de RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Transgenes , Northern Blotting , Genes de Plantas/genética , Íntrons/genética , Precursores de RNA/genética , Processamento de RNA , RNA Interferente Pequeno/genética , Análise de Sequência de RNA , Tabaco/genética , Tabaco/metabolismo , Transgenes/genética
14.
Methods Mol Biol ; 1870: 179-187, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30539555

RESUMO

Immuno-northern blotting is a method for detecting modified RNAs using gel separation and specific antibodies to modified nucleosides. This method was developed by combining two commonly used molecular biology techniques: western blotting and northern blotting. In this method, urea-polyacrylamide (or agarose) gel-separated RNAs are transferred to positively charged nylon membrane and then immune detection is performed with specific antibodies to modified nucleosides: such as 1-methyladenosine, N6-methyladenosine, and pseudouridine. This highly sensitive and relatively simple method, which uses widely available laboratory equipment, enables small laboratories to compare the abundance of modified nucleic acids across samples.


Assuntos
Anticorpos , Northern Blotting , Nucleosídeos , RNA , Adenosina/análogos & derivados , Northern Blotting/métodos , RNA/química , RNA/genética
15.
FASEB J ; 33(1): 808-820, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30063439

RESUMO

Carboxypeptidase E (CPE), an exopeptidase involved in proneuropeptide processing, is also a neurotrophic factor, named neurotrophic factor-α1 (NF-α1) and has important roles in neuroprotection, stem cell differentiation, and neurite outgrowth, independent of enzymatic activity. Additionally, an N-terminal-truncated CPE/NF-α1 variant, (CPE/NF-α1)-ΔN, proposed from bioinformatic analysis of GenBank (National Center for Biotechnology Information, Bethesda, MD, USA) DNA sequences and encoding a 40-kDa protein, has been found to be exclusively expressed in embryonic neurons. To investigate the function of (CPE/NF-α1)-ΔN in neurodevelopment, we first cloned (CPE/NF-α1)-ΔN transcripts from an embryonic mouse brain. A rapid amplification of cDNA ends assay, DNA sequencing, and Northern blot revealed 1.9- and 1.73-kb transcripts, which encoded 47- and 40-kDa (CPE/NF-α1)-ΔN proteins, respectively. Those proteins were expressed in embryonic mouse brain. Expression of the 2 (CPE/NF-α1)-ΔN mRNAs surged at embryonic d 10.5, correlating with the time of neurogenesis in the developing brain and also at postnatal d 1. HT22 cells, a mouse hippocampal cell line, transduced with 40 kDa (CPE/NF-α1)-ΔN up-regulated expression of genes involved in embryonic neurodevelopment: insulin-like growth factor binding protein 2 ( IGFBP2), death-associated protein 1, and ephrin A1, which regulate proliferation, programmed cell death, and neuronal migration, respectively. HT22 cells and embryonic cortical neurons overexpressing 40 kDa (CPE/NF-α1)-ΔN exhibited enhanced proliferation, which was inhibited by IGFBP2 short interfering RNA treatment. Thus, 40 kDa (CPE/NF-α1)-ΔN has an important, enzymatically independent role in the regulation of genes critical for neurodevelopment.-Xiao, L., Yang, X., Sharma, V. K., Loh, Y. P. Cloning, gene regulation, and neuronal proliferation functions of novel N-terminal-truncated carboxypeptidase E/neurotrophic factor-αl variants in embryonic mouse brain.


Assuntos
Encéfalo/embriologia , Carboxipeptidase H/metabolismo , Proliferação de Células , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurônios/citologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Encéfalo/citologia , Encéfalo/metabolismo , Carboxipeptidase H/genética , Linhagem Celular , Concentração de Íons de Hidrogênio , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Regulação para Cima
16.
Methods ; 155: 124-130, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30448478

RESUMO

Post-transcriptional RNA metabolic pathways play important roles in permitting prokaryotes to operate under a variety of environmental conditions. Although significant progress has been made during the last decade in deciphering RNA processing pathways in a number of bacteria, a complete understanding of post-transcriptional RNA metabolism in any single microorganism is far from reality. Here we describe multiple experimental approaches that can be used to study mRNA stability, tRNA and rRNA processing, sRNA metabolism, and polyadenylation in prokaryotes. The methods described here can be readily utilized in both Gram-negative and Gram-positive bacteria with simple modifications.


Assuntos
Escherichia coli/genética , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Mensageiro/genética , Análise de Sequência de DNA/métodos , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Eletroforese em Gel de Gradiente Desnaturante , Desoxirribonuclease I/química , Escherichia coli/metabolismo , Meia-Vida , Poliadenilação , Estabilidade de RNA , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo
17.
Cold Spring Harb Protoc ; 2018(11)2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30385675

RESUMO

Purification of high-quality total RNA from specimens is essential for many molecular techniques. In tardigrades (water bears), disruption of the cuticle is an important step in obtaining a good yield that is representative of all tissues of the animal. As with all single-stranded RNA methods, sterile technique, proper storage conditions, and handling are required for maintaining the quality and integrity of the material. This procedure will result in high-quality total RNA suitable for downstream applications such as cDNA synthesis, reverse transcriptase-polymerase chain reaction (RT-PCR), RNAseq library generation, and northern blots.


Assuntos
Northern Blotting/métodos , DNA Complementar/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tardígrados/genética , Animais , RNA/genética
18.
Adv Exp Med Biol ; 1087: 3-14, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30259353

RESUMO

Circular RNAs (cirRNAs) are long, noncoding endogenous RNA molecules and covalently closed continuous loop without 5'-3' polarity and polyadenylated tail which are largely concentrated in the nucleus. CirRNA regulates gene expression by modulating microRNAs and functions as potential biomarker. CirRNAs can translate in vivo to link between their expression and disease. They are resistant to RNA exonuclease and can convert to the linear RNA by microRNA which can then act as competitor to endogenous RNA. This chapter summarizes the evolutionary conservation and expression of cirRNAs, their identification, highlighting various computational approaches on cirRNA, and translation with a focus on the breakthroughs and the challenges in this new field.


Assuntos
RNA/genética , Northern Blotting , Biologia Computacional , Regulação da Expressão Gênica/genética , Hibridização in Situ Fluorescente , MicroRNAs/genética , Reação em Cadeia da Polimerase/métodos , Biossíntese de Proteínas , RNA/análise , RNA/química , Processamento Pós-Transcricional do RNA , RNA Longo não Codificante/análise , RNA Longo não Codificante/química , RNA Longo não Codificante/genética
19.
RNA ; 24(12): 1871-1877, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30201850

RESUMO

Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive 32P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than 32P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.


Assuntos
Northern Blotting/métodos , Corantes Fluorescentes/química , Hibridização de Ácido Nucleico/métodos , RNA/isolamento & purificação , Sondas de DNA/química , RNA/química , Sondas RNA/química
20.
Plant Cell Environ ; 41(12): 2844-2857, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30103284

RESUMO

Beta vulgaris (sugar beet) is one of the most important industrial crops. Screening of a cDNA library for sugar beet genes able to confer cold tolerance upon overexpression in yeast identified a novel aquaporin, which we named BvCOLD1. The amino acid sequence of BvCOLD1 indicated that an acidic protein (pI 5.18) is similar to tonoplast intrinsic protein aquaporins. RNA expression analysis indicated that BvCOLD1 is expressed in all sugar beet organs. Confocal microscopy of a green fluorescent protein-tagged version localized BvCOLD1 in the endoplasmic reticulum in yeast and in plant cells. Experiments in yeast showed that BvCOLD1 has an important role in transporting several molecules, among them is boron, one of the most limiting micronutrients for sugar beet cultivation. Transgenic Arabidopsis thaliana plants overexpressing BvCOLD1 showed enhanced tolerance to cold, to different abiotic stresses, and to boron deficiency at different developmental stages. Searches in databases only retrieved BvCOLD1 orthologues in genomes from the Chenopodioideae, a subfamily of the Amaranthaceae family that includes the closely related crop Spinacea oleracea and halotolerant plants such as Salicornia herbacea or Suaeda glauca. Orthologues share a conserved sequence in the carboxy terminus, not present in other aquaporins, which is required for the functionality of the protein.


Assuntos
Aquaporinas/metabolismo , Beta vulgaris/metabolismo , Boro/metabolismo , Proteínas de Plantas/metabolismo , Aquaporinas/genética , Aquaporinas/fisiologia , Arabidopsis , Beta vulgaris/genética , Beta vulgaris/fisiologia , Northern Blotting , Temperatura Baixa , Retículo Endoplasmático/metabolismo , Homeostase , Microscopia Confocal , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Estresse Fisiológico , Tabaco
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