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1.
Nat Commun ; 11(1): 4104, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796835

RESUMO

Transfer RNAs (tRNA) are quintessential in deciphering the genetic code; disseminating nucleic acid triplets into correct amino acid identity. While this decoding function is clear, an emerging theme is that tRNA abundance and functionality can powerfully impact protein production rate, folding, activity, and messenger RNA stability. Importantly, however, the expression pattern of tRNAs is obliquely known. Here we present Quantitative Mature tRNA sequencing (QuantM-tRNA seq), a technique to monitor tRNA abundance and sequence variants secondary to RNA modifications. With QuantM-tRNA seq, we assess the tRNA transcriptome in mammalian tissues. We observe dramatic distinctions in isodecoder expression and known tRNA modifications between tissues. Remarkably, despite dramatic changes in tRNA isodecoder gene expression, the overall anticodon pool of each tRNA family is similar across tissues. These findings suggest that while anticodon pools appear to be buffered via an unknown mechanism, underlying transcriptomic and epitranscriptomic differences suggest a more complex tRNA regulatory landscape.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA de Transferência/metabolismo , Animais , Anticódon/genética , Northern Blotting , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , RNA de Transferência/genética
2.
J Vis Exp ; (161)2020 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-32716394

RESUMO

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.


Assuntos
Northern Blotting/métodos , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Plantas/genética , Animais , Sequência de Bases , RNA Helicases DEAD-box , Expressão Gênica , RNA Mensageiro/genética , RNA de Plantas/genética , Ribonuclease III
3.
PLoS One ; 15(4): e0230958, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32294092

RESUMO

Soil salinization is a serious problem for cultivation of rice, as among cereals rice is the most salt sensitive crop, and more than 40% of the total agricultural land amounting to approximately 80 million ha the world over is salt affected. Salinity affects a plant in a varieties of ways, including ion toxicity, osmotic stress and oxidative damage. Since miRNAs occupy the top place in biochemical events determining a trait, understanding their role in salt tolerance is highly desirable, which may allow introduction of the trait in the rice cultivars of choice through biotechnological interventions. High throughput sequencing of sRNAs in the root and shoot tissues of the seedlings of the control and NaCl treated Pokkali, a salt-tolerant rice variety, identified 75 conserved miRNAs and mapped 200 sRNAs to the rice genome as novel miRNAs. Expression of nine novel miRNAs and two conserved miRNAs were confirmed by Northern blotting. Several of both conserved and novel miRNAs that expressed differentially in root and/or shoot tissues targeted transcription factors like AP2/EREBP domain protein, ARF, NAC, MYB, NF-YA, HD-Zip III, TCP and SBP reported to be involved in salt tolerance or in abiotic stress tolerance in general. Most of the novel miRNAs expressed in the salt tolerant wild rice Oryza coarctata, suggesting conservation of miRNAs in taxonomically related species. One of the novel miRNAs, osa-miR12477, also targeted L-ascorbate oxidase (LAO), indicating build-up of oxidative stress in the plant upon salt treatment, which was confirmed by DAB staining. Thus, salt tolerance might involve miRNA-mediated regulation of 1) cellular abundance of the hormone signaling components like EREBP and ARF, 2) synthesis of abiotic stress related transcription factors, and 3) antioxidative component like LAO for mitigation of oxidative damage. The study clearly indicated importance of osa-miR12477 regulated expression of LAO in salt tolerance in the plant.


Assuntos
MicroRNAs/genética , Oryza/genética , Tolerância ao Sal/genética , Northern Blotting/métodos , Regulação da Expressão Gênica de Plantas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Estresse Oxidativo/genética , Salinidade , Plântula/genética , Estresse Fisiológico/genética , Fatores de Transcrição/genética
4.
Development ; 147(9)2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32273274

RESUMO

MicroRNAs (miRNAs) are short (∼22 nt) single-stranded non-coding RNAs that regulate gene expression at the post-transcriptional level. Over recent years, many studies have extensively characterized the involvement of miRNA-mediated regulation in neurogenesis and brain development. However, a comprehensive catalog of cortical miRNAs expressed in a cell-specific manner in progenitor types of the developing mammalian cortex is still missing. Overcoming this limitation, here we exploited a double reporter mouse line previously validated by our group to allow the identification of the transcriptional signature of neurogenic commitment and provide the field with the complete atlas of miRNA expression in proliferating neural stem cells, neurogenic progenitors and newborn neurons during corticogenesis. By extending the currently known list of miRNAs expressed in the mouse brain by over twofold, our study highlights the power of cell type-specific analyses for the detection of transcripts that would otherwise be diluted out when studying bulk tissues. We further exploited our data by predicting putative miRNAs and validated the power of our approach by providing evidence for the involvement of miR-486 in brain development.


Assuntos
MicroRNAs/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Animais , Northern Blotting , Biologia Computacional/métodos , Eletroporação , Feminino , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neurogênese/genética , Neurogênese/fisiologia
5.
Adv Wound Care (New Rochelle) ; 9(4): 145-160, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32117579

RESUMO

Objective: Insufficient knowledge about the molecular pathology of diabetic foot ulcer (DFU) impedes the development of effective wound treatment. Circular RNAs (circRNAs) are a novel class of RNA recently discovered to be widely expressed and have important biological functions; however, their role in skin wound healing remains largely unexplored. In this study, we investigated the role of circRNAs in DFU. Approach: CircRNA expression was profiled in normal wounds (NWs) and DFUs by microarray analysis, and hsa_circ_0084443 was identified as differentially expressed. The circularity and subcellular localization of hsa_circ_0084443 were characterized by northern blotting, real-time PCR, and fluorescence in situ hybridization. Cell migration, cell growth, and the transcriptome of human primary keratinocytes were analyzed after overexpression or RNA interference of hsa_circ_0084443. Results: hsa_circ_0084443 is downregulated in NWs compared with intact skin, and its level is higher in DFUs than NWs. We confirmed its circularity and presence in the cytoplasm of human epidermal keratinocytes. We showed that hsa_circ_0084443 reduced motility while enhancing the growth of keratinocytes. Furthermore, we identified a gene network with the potential to mediate the biological effect of hsa_circ_0084443. Innovation: CircRNAs have a functional role and a potential clinical significance in skin wound healing. Conclusions: We identified hsa_circ_0084443, a circRNA downregulated during NW healing, as a negative regulator of keratinocyte migration. Higher levels of hsa_circ_0084443 were detected in DFU samples, suggesting that it plays a role in pathology. These findings pave the way to understanding the functional role of circRNAs in human skin wound healing.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Pé Diabético/genética , Queratinócitos/metabolismo , RNA Circular/genética , Regulação para Cima/genética , Cicatrização/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Northern Blotting , Estudos de Coortes , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , RNA Circular/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma
6.
J Diabetes Res ; 2020: 2057187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32083134

RESUMO

We report here the clinical, genetic, and molecular characteristics of type 2 diabetes in a Chinese family. There are differences in the severity and age of onset in diabetes among these families. By molecular analysis of the complete mitochondrial genome in this family, we identified the homoplasmic m.15897G>A mutation underwent sequence analysis of whole mitochondrial DNA genome, which localized at conventional position ten of tRNAThr, and distinct sets of mtDNA polymorphisms belonging to haplogroup D4b1. This mutation has been implicated to be important for tRNA identity and stability. Using cybrid cell models, the decreased efficiency of mitochondrial tRNAThr levels caused by the m.15897G>A mutation results in respiratory deficiency, protein synthesis and assembly, mitochondrial ATP synthesis, and mitochondrial membrane potential. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cell lines. These data provide a direct evidence that a novel tRNA mutation was associated with T2DM. Thus, our findings provide a new insight into the understanding of pathophysiology of maternally inherited diabetes.


Assuntos
Diabetes Mellitus Tipo 2/genética , RNA de Transferência de Treonina/genética , Adulto , Idoso , Grupo com Ancestrais do Continente Asiático/genética , Northern Blotting , Western Blotting , China , Diabetes Mellitus Tipo 2/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Genoma Mitocondrial , Humanos , Masculino , Herança Materna , Potencial da Membrana Mitocondrial/genética , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Linhagem , Filogenia , Mutação Puntual , Espécies Reativas de Oxigênio/metabolismo
7.
Appl Microbiol Biotechnol ; 104(2): 833-852, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31848654

RESUMO

Bacillus pumilus, an endospore-forming soil bacterium, produces a wide array of extracellular proteins, such as proteases, which are already applied in the chemical, detergent and leather industries. Small noncoding regulatory RNAs (sRNAs) in bacteria are important RNA regulators that act in response to various environmental signals. Here, an RNA-seq-based transcriptome analysis was applied to B. pumilus SCU11, a strain that produces extracellular alkaline protease, across various growth phases of the protease fermentation process. Through bioinformatics screening of the sequencing data and visual inspection, 84 putative regulatory sRNAs were identified in B. pumilus, including 21 antisense sRNAs and 63 sRNAs in intergenic regions. We experimentally validated the expression of 48 intergenic sRNAs by quantitative RT-PCR (qRT-PCR). Meanwhile, the expression of 6 novel sRNAs was confirmed by northern blotting, and the expression profiles of 5 sRNAs showed close correlation with the growth phase. We revealed that the sRNA Bpsr137 was involved in flagellum and biofilm formation in B. pumilus. The identification of a global set of sRNAs increases the inventory of regulatory sRNAs in Bacillus and implies the important regulatory roles of sRNA in B. pumilus. These findings will contribute another dimension to the optimization of crucial metabolic activities of B. pumilus during a productive fermentation process.


Assuntos
Bacillus pumilus/crescimento & desenvolvimento , Bacillus pumilus/genética , Peptídeo Hidrolases/metabolismo , Pequeno RNA não Traduzido/biossíntese , Bacillus pumilus/metabolismo , Northern Blotting , Biologia Computacional , Fermentação , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Pequeno RNA não Traduzido/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA
8.
Methods Mol Biol ; 2062: 83-103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31768973

RESUMO

Over the past decade a plethora of noncoding RNAs (ncRNAs) have been identified, initiating an explosion in RNA research. Although RNA sequencing methods provide unsurpassed insights into ncRNA distribution and expression, detailed information on structure and processing are harder to extract from sequence data. In contrast, northern blotting methods provide uniquely detailed insights into complex RNA populations but are rarely employed outside specialist RNA research groups. Such techniques are generally considered difficult for nonspecialists, which is unfortunate as substantial technical advances in the past few decades have solved the major challenges. Here we present simple, reproducible and highly robust protocols for separating glyoxylated RNA on agarose gels and heat denatured RNA on polyacrylamide-urea gels using standard laboratory electrophoresis equipment. We also provide reliable transfer and hybridization protocols that do not require optimization for most applications. Together, these should allow any molecular biology lab to elucidate the structure and processing of ncRNAs of interest.


Assuntos
Northern Blotting/métodos , Exossomos/genética , RNA não Traduzido/genética , Resinas Acrílicas/química , Eletroforese em Gel de Ágar/métodos , Hibridização de Ácido Nucleico/métodos , Análise de Sequência de RNA/métodos
9.
Methods Mol Biol ; 2052: 205-218, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31452164

RESUMO

MicroRNAs (miRNAs) represent a subclass of endogenous small noncoding RNAs that have been identified in both mammalian and nonmammalian cells. miRNAs are an essential part of the complex regulatory networks that control numerous biological processes and may play an important role in host defense and/or microbial offense during host-parasite interactions. Here, several methodologies to explore the role for miRNAs in host-parasite interactions are briefly summarized, including the detection, quantification, and intracellular localization of miRNAs, identification and validation of miRNA targets, and functional manipulation of specific miRNAs.


Assuntos
Cryptosporidium/genética , Interações Hospedeiro-Parasita/genética , MicroRNAs/genética , Northern Blotting/métodos , Western Blotting/métodos , Linhagem Celular , Cryptosporidium/patogenicidade , Bases de Dados Genéticas , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Genes Reporter , Humanos , Hibridização In Situ/métodos , Luciferases/genética , Luciferases/metabolismo , MicroRNAs/isolamento & purificação , MicroRNAs/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fluxo de Trabalho
10.
EMBO Mol Med ; 11(12): e10835, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31709724

RESUMO

Aerobic glycolysis is a hallmark of metabolic reprogramming in tumor progression. However, the mechanisms regulating glycolytic gene expression remain elusive in neuroblastoma (NB), the most common extracranial malignancy in childhood. Herein, we identify that CUT-like homeobox 1 (CUX1) and CUX1-generated circular RNA (circ-CUX1) contribute to aerobic glycolysis and NB progression. Mechanistically, p110 CUX1, a transcription factor generated by proteolytic processing of p200 CUX1, promotes the expression of enolase 1, glucose-6-phosphate isomerase, and phosphoglycerate kinase 1, while circ-CUX1 binds to EWS RNA-binding protein 1 (EWSR1) to facilitate its interaction with MYC-associated zinc finger protein (MAZ), resulting in transactivation of MAZ and transcriptional alteration of CUX1 and other genes associated with tumor progression. Administration of an inhibitory peptide blocking circ-CUX1-EWSR1 interaction or lentivirus mediating circ-CUX1 knockdown suppresses aerobic glycolysis, growth, and aggressiveness of NB cells. In clinical NB cases, CUX1 is an independent prognostic factor for unfavorable outcome, and patients with high circ-CUX1 expression have lower survival probability. These results indicate circ-CUX1/EWSR1/MAZ axis as a therapeutic target for aerobic glycolysis and NB progression.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Glicólise , Células HEK293 , Células HeLa , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Imunoprecipitação , Técnicas In Vitro , Espectrometria de Massas , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuroblastoma , Células PC-3 , Proteína EWS de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Fatores de Transcrição/genética
11.
mBio ; 10(4)2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363026

RESUMO

Endogenous retroviruses (ERVs) are transposable elements that cause host genome instability and usually play deleterious roles in disease such as tumorigenesis. Recent advances also suggest that this "enemy within" may encode a viral mimic to induce antiviral immune responses through viral sensors. Here, through whole-genome transcriptome analysis with RNA sequencing (RNA-Seq), we discovered that a full-length ERV-derived long noncoding RNA (lncRNA), designated lnc-EPAV (ERV-derived lncRNA positively regulates antiviral responses), was a positive regulator of NF-κB signaling. lnc-EPAV expression was rapidly upregulated by viral RNA mimics or RNA viruses to facilitate the expression of RELA, an NF-κB subunit that plays a crucial role in antiviral responses. Transcriptome analysis of lnc-EPAV-silenced macrophages showed that lnc-EPAV was critical for RELA target gene expression and innate immune responses. Consistently, lnc-EPAV-deficient mice exhibited reduced expression of type I interferons (IFNs) and, consequently, increased viral loads and mortality following lethal RNA virus infection. Mechanistically, lnc-EPAV promoted expression of RELA by competitively binding to and displacing SFPQ, a transcriptional repressor of Rela Altogether, our work demonstrates an alternative mechanism by which ERVs regulate antiviral immune responses.IMPORTANCE Endogenous retroviruses are transposable genetic elements comprising 8% to 10% of the human and mouse genomes. Although most ERVs have been inactivated due to deleterious mutations, some are still transcribed. However, the biological functions of transcribed ERVs are largely unknown. Here, we identified a full-length ERV-derived lncRNA, designated lnc-EPAV, as a positive regulator of host innate immune responses. We found that silencing lnc-EPAV impaired virus-induced cytokine production, resulting in increased viral replication in cells. The lnc-EPAV-deficient mice exhibited enhanced susceptibility to viral challenge. We also found that lnc-EPAV regulated expression of RELA, an NF-κB subunit that plays a critical role in antiviral responses. ERV-derived lncRNA coordinated with a transcription repressor, SFPQ, to control Rela transcription. Our report provides new insights into the previously unrecognized immune gene regulatory mechanism of ERV-derived lncRNAs.


Assuntos
Imunidade Inata/fisiologia , RNA Longo não Codificante/genética , Fator de Transcrição RelA/metabolismo , Animais , Northern Blotting , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Imunidade Inata/genética , Immunoblotting , Imunoprecipitação , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Fator de Transcrição RelA/genética , Células Vero
12.
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
13.
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
14.
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
15.
Neuroscience ; 411: 23-36, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31128160

RESUMO

The sphenopalatine ganglion (SPG) is a gathering of the cell bodies of parasympathetic fibers that dominate the nasal gland, lacrimal gland and cerebral blood vessels. The SPG controls nasal secretions, tears, and the dilation of cerebral blood vessels. However, it is unclear how serotonin regulates SPG functions. In this study, we investigated the expression of genes involved in the serotonergic system in the mouse SPG. We examined the mRNA expression levels of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3A, 5-HT3B, 5-HT4, 5-HT5A, 5-HT5B, 5-HT6 and 5-HT7 receptors, as well as serotonin transporter, tryptophan hydroxylases 1 and 2, and L-amino acid decarboxylase (AADC) by RT-PCR. It revealed that the 5-HT3A and 5-HT3B ionotropic receptors and AADC were likely to be highly expressed in the SPG, as measured by RT-PCR. We next performed in situ hybridization on the SPG to examine the expression of these three genes at the cellular level after validating the specificity of each cRNA probe by northern blotting. The 5-HT3A receptor, 5-HT3B receptor, and AADC were expressed in 96.5% ±â€¯1.0%, 29.7% ±â€¯10.7%, and 57.4% ±â€¯2.9% of neuronal cell bodies in the SPG, respectively, indicating that the 5-HT3A receptor was virtually expressed in all SPG neurons. Our results on the expression of these critical serotonin system genes in the parasympathetic SPG provide insight into the pathogenetics of rhinitis, conjunctivitis and headache. Furthermore, our findings suggest that targeting the 5-HT3A receptor might have therapeutic potential in the treatment of these ailments.


Assuntos
Gânglios Parassimpáticos/metabolismo , Neurônios/metabolismo , Receptores de Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Triptofano Hidroxilase/metabolismo , Animais , Northern Blotting , Hibridização In Situ , Masculino , Camundongos , Receptores de Serotonina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Triptofano Hidroxilase/genética
16.
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
17.
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
18.
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
19.
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
20.
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
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