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1.
Medicine (Baltimore) ; 98(42): e17601, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31626137

RESUMO

BACKGROUND: Lung adenocarcinoma (LA) is a most common form of non-small cell lung cancer (NSCLC). To date, there are still no effective early diagnosis methods for patients to be cured in time. Noncoding RNA plays an important role in oncogenesis and tumor development. The expression profile of circular RNA (circRNA) in peripheral whole blood (PWB) of LA has not been systematically investigated. In this study, we identified the differentially expressed (DE) circRNAs in PWB of LA by high-throughput sequencing. METHODS: Five paired LA and normal participants PWB samples were chosen to investigate the expression profile of circRNAs by high-throughput sequencing. Twenty LA and 10 normal controls PWB samples were subjected to reverse-transcription polymerase chain reaction (RT-PCR) for validation of circRNAs expression profile. Gene Ontology (GO) functional analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and circRNA-miRNA network analysis was also performed to predict the function of circRNAs in PWB. RESULTS: A total of 10566 circRNAs were identified and annotated, most of the circRNAs were exonic (78.14%). Statistical analysis revealed 4390 DE circRNAs, in which were 3009 upregulated circRNAs and1381downregulated circRNAs in LA. RT-PCR results showed that circRNA expression in LA was higher than that in controls. GO functional analysis, KEGG pathway analysis, and circRNA-miRNA network analysis all showed that circRNAs correlated with tumor development and progression to a certain degree. The current study is the first to systematically characterize and annotate circRNA expression in PWB of LA. Some host genes of the DE circRNAs were involved in tumor signaling pathway and had complicated correlations with tumor related miRNAs, indicating that circRNAs might involve in development and progression of LA. CONCLUSIONS: Our study revealed that circRNAs were abnormally expressed in PWB of LA, which might offer potential targets for the early diagnosis of the disease and new genetic insights into LA.


Assuntos
Adenocarcinoma de Pulmão/genética , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Adenocarcinoma de Pulmão/sangue , Perfilação da Expressão Gênica/métodos , Humanos , RNA/biossíntese , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Regulação para Cima
2.
Phys Rev Lett ; 123(3): 038101, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31386470

RESUMO

Synthesis of biopolymers such as DNA, RNA, and proteins are biophysical processes aided by enzymes. The performance of these enzymes is usually characterized in terms of their average error rate and speed. However, because of thermal fluctuations in these single-molecule processes, both error and speed are inherently stochastic quantities. In this Letter, we study fluctuations of error and speed in biopolymer synthesis and show that they are in general correlated. This means that, under equal conditions, polymers that are synthesized faster due to a fluctuation tend to have either better or worse errors than the average. The error-correction mechanism implemented by the enzyme determines which of the two cases holds. For example, discrimination in the forward reaction rates tends to grant smaller errors to polymers with faster synthesis. The opposite occurs for discrimination in monomer rejection rates. Our results provide an experimentally feasible way to identify error-correction mechanisms by measuring the error-speed correlations.


Assuntos
Biopolímeros/biossíntese , Enzimas/química , Enzimas/metabolismo , Biopolímeros/química , DNA/biossíntese , DNA/química , Humanos , Modelos Biológicos , Modelos Químicos , RNA/biossíntese , RNA/química
3.
Nat Commun ; 10(1): 2948, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270316

RESUMO

CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Biblioteca Gênica , Técnicas Genéticas , RNA/biossíntese , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA/genética , Endonucleases/metabolismo , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo
4.
J Clin Pathol ; 72(8): 513-519, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154423

RESUMO

The importance of circular RNAs (circRNAs) in pathological processes like cancer is evident. Among the circRNAs, recent studies have brought circPVT1 under focus as the most potent oncogenic non-coding RNA. Recent studies on various aspects of circPVT1, including its biogenesis, molecular alteration and its probable role in oncogenesis, have been conducted for research and clinical interest. In this review, a first attempt has been made to summarise the available data on circPVT1 from PubMed and other relevant databases with special emphasis on its role in development, progression and prognosis of various malignant conditions. CircPVT1 is derived from the same genetic locus encoding for long non-coding RNA lncPVT1; however, existing literature suggested circPVT1 and lncPVT1 are transcripted independently by different promoters. The interaction between circRNA and microRNA has been highlighted in majority of the few malignancies in which circPVT1 was studied. Besides its importance in diagnostic and prognostic procedures, circPVT1 seemed to have huge therapeutic potential as evident from differential drug response of cancer cell line as well as primary tumors depending on expression level of the candidate. circPVT1 in cancer therapeutics might be promising as a biomarker to make the existing treatment protocol more effective and also as potential target for designing novel therapeutic intervention.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias/genética , RNA/genética , Animais , Biomarcadores Tumorais/biossíntese , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas de Diagnóstico Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Valor Preditivo dos Testes , Prognóstico , RNA/biossíntese , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/metabolismo
5.
Biomed Res Int ; 2019: 3842312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058188

RESUMO

There are about 1-2 million follicles presented in the ovary at birth, while only around 1000 primordial follicles are left at menopause. The ovarian function also decreases in parallel with aging. Folliculogenesis is vital for ovarian function, no matter the synthesis of female hormones or ovulation, yet the mechanisms for its changing with increasing age are not fully understood. Early follicle growth up to the large preantral stage is independent of gonadotropins in rodents and relies on intraovarian factors. To further understand the age-related molecular changes in the process of folliculogenesis, we performed microarray gene expression profile analysis using total RNA extracted from young (9 weeks old) and old (32 weeks old) mouse ovarian secondary follicles. The results of our current microarray study revealed that there were 371 (≥2-fold, q-value ≤0.05) genes differentially expressed in which 174 genes were upregulated and 197 genes were downregulated in old mouse ovarian secondary follicles compared to young mouse ovarian secondary follicles. The gene ontology and KEGG pathway analysis of differentially expressed genes uncovered critical biological functions such as immune system process, aging, transcription, DNA replication, DNA repair, protein stabilization, and apoptotic process were affected in the process of aging. The considerable changes in gene expression profile may have an adverse influence on follicle quality and folliculogenesis. Our study provided information on the processes that may contribute to age-related decline in ovarian function.


Assuntos
Envelhecimento/genética , Folículo Ovariano/crescimento & desenvolvimento , Ovário/crescimento & desenvolvimento , RNA/genética , Animais , Reparo do DNA/genética , Replicação do DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Menopausa/genética , Camundongos , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , Ovulação/genética , RNA/biossíntese , Transcriptoma/genética
6.
Mol Biotechnol ; 61(7): 469-476, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30868354

RESUMO

RNA elements such as catalytic RNA, riboswitch, microRNA, and long non-coding RNA perform a major role in cellular processes. A complete understanding of cellular processes is impossible without knowing the structure-function relationship of participating RNA molecules that ultimately requires large quantities of pure RNAs. Thus, structural/functional analyses of emerging RNAs necessitate revised protocols for improved RNA quantity and quality. Here we present a modified in vitro transcription protocol to enhance ribozyme cleaving efficiency and RNA yield by working on two variables, i.e., incubation temperature and limiting GTPs. Following an improved RNA synthesis, the target RNA is purified from transcription mixture components through denaturing size-exclusion chromatography. The protocol confirms that cyclic elevated incubation temperatures during transcription and increased concentrations of GTPs improve the production rate of RNA. Our modified in vitro transcription method improves the ribozyme cleaving efficiency and targets RNA yield by four- to fivefold that can benefit almost any RNA-related study from protein-RNA interaction analysis to crystallography.


Assuntos
Técnicas In Vitro/normas , RNA Catalítico , RNA/biossíntese , Transcrição Genética , Cromatografia em Gel , Guanosina Trifosfato , RNA/química , Temperatura Ambiente
7.
Gene ; 701: 139-145, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30914324

RESUMO

CircRNAs (Circular RNAs) are believed to be involved in a multitude of biological processes and can play crucial roles as miRNA (microRNA) sponges. The aim of this study was to explore the relationship between circRNAs and ischemic stroke induced by middle cerebral artery occlusion (MCAO) in rats. The expression profiles of circRNAs in brain tissues were screened by high-throughput sequencing (HTS) to identify novel differentially expressed circRNAs (DECs). Quantitative real-time (qRT)-PCR was used to confirm circRNA expression data arising from HTS. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to investigate mRNA function and to understand the characteristics of circRNAs. Finally, we created a circRNA-miRNA network. In total, 14,694 DECs, from 6 brain tissues, were screened; of these, 87 DECs showed a significant fold change >2 (p < 0.05). CircRNAs showing significant upregulation included circRNA.17737, circRNA.8828 and circRNA.14479, while circRNA.1059, circRNA.9967 and circRNA.6952 were significant downregulation. The results were in accordance with HTS, showing that the HTS produced reliable data. Herein, we explored the relationship between circRNAs and ischemic stroke induced by MCAO in rats. Our data provide a new and better understanding of the molecular mechanisms associated with ischemic stroke. The precise role of DECs associated with MCAO and target miRNAs should be validated in further studies.


Assuntos
Encéfalo , Infarto da Artéria Cerebral Média , RNA , Acidente Vascular Cerebral , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , RNA/biossíntese , RNA/genética , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia
8.
Turk J Med Sci ; 49(2): 687-695, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30866608

RESUMO

Background/aim: Gastric cancer (GC) is one of the major causes of cancer mortality worldwide. As a novel type of endogenous noncoding RNAs, circular RNAs (circRNAs) are formed by a covalent link between 5' and 3' ends. They are very stable and abundant in eukaryotes. As there were no reported studies on the expression profiles of circular RNA ITCH (cir-ITCH) and circHIPK3 in GC, in the current study, we aimed to delineate the expression profiles and clinicopathological relevance of these two circRNAs in GC tissues compared to their paired adjacent noncancerous tissues. Materials and methods: Quantitative real-time polymerase chain reaction was performed to evaluate cir_ITCH and circHIPK3 expression in 30 paired gastric cancer tissues. The clinicopathological relevance of these two circular RNAs' expression levels with gastric cancer was further examined. Results: Our results showed that the expression of cir_ITCH and circHIPK3 were significantly downregulated in GC tumoral tissues compared with their paired adjacent nonneoplastic counterparts. Further analyses showed that cir_ITCH and circHIPK3 expression levels were related with numerous clinicopathological features of tumoral tissues. Conclusion: Cir_ITCH and circHIPK3 may have imperative roles in GC and serve in the future as potential prognostic biomarkers in GC.


Assuntos
Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , RNA/genética , Proteínas Repressoras/genética , Neoplasias Gástricas/genética , Ubiquitina-Proteína Ligases/genética , Idoso , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , RNA/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Neoplasias Gástricas/patologia
9.
Nat Commun ; 10(1): 1185, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862852

RESUMO

Cellular products derived from the activity of DNA, RNA, and protein synthesis collectively control cell identity and function. Yet there is little information on how these three biosynthesis activities are coordinated during transient and sparse cellular processes, such as activation and differentiation. Here, we describe Simultaneous Overview of tri-Molecule Biosynthesis (SOM3B), a molecular labeling and simultaneous detection strategy to quantify DNA, RNA, and protein synthesis in individual cells. Comprehensive interrogation of biosynthesis activities during transient cell states, such as progression through cell cycle or cellular differentiation, is achieved by partnering SOM3B with parallel quantification of select biomolecules with conjugated antibody reagents. Here, we investigate differential de novo DNA, RNA, and protein synthesis dynamics in transformed human cell lines, primary activated human immune cells, and across the healthy human hematopoietic continuum, all at a single-cell resolution.


Assuntos
DNA/biossíntese , Biossíntese de Proteínas , RNA/biossíntese , Análise de Célula Única/métodos , Medula Óssea/metabolismo , Ciclo Celular , Células HEK293 , Células HeLa , Voluntários Saudáveis , Humanos , Células Jurkat , Leucócitos Mononucleares , Cultura Primária de Células , Coloração e Rotulagem/métodos
10.
Mol Cell ; 74(1): 212-222.e5, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30795893

RESUMO

Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here we present a multiplexed, sequential imaging approach (Hi-M) that permits simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome unpairing during awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understanding the mechanisms and consequences of the 4D organization of the genome.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromossomos de Insetos/genética , Drosophila melanogaster/genética , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microscopia de Fluorescência/métodos , RNA/genética , Análise de Célula Única/métodos , Transcrição Genética , Ativação Transcricional , Animais , Ciclo Celular/genética , Cromatina/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização in Situ Fluorescente , RNA/biossíntese
11.
Biomed Pharmacother ; 112: 108611, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30797148

RESUMO

Circular RNAs (circRNAs) are a novel class of non-coding RNAs with distinct properties and diverse physiological and pathological functions. However, the functions of circRNAs in colorectal cancer (CRC) remain elusive. This study aimed to investigate the functional roles of circVAPA in CRC. High-throughput RNA sequencing was performed in 4 paired CRC tissues, and circVAPA (hsa_circ_0006990), was identified as a potential functional circRNA. Using quantitative real-time polymerase chain reaction (qRT-PCR), circVAPA was found to be up-regulated in CRC patients' tissues and plasma. Furthermore, circVAPA level was associated with unfavorable clinicopathologic features in CRC. The area under curve (AUC) of ROC was 0.724, suggesting that plasma level of circVAPA could serve as a promising biomarker for CRC detection. Sanger sequencing confirmed the back-splice junction sequences of circVAPA. Actinomycin D and RNase R treatments suggested that circVAPA was highly stable compared with its linear counterpart, and qRT-PCR for the circVAPA level in nuclear and cytoplasmic fractions indicated that circVAPA was predominantly localized in the cytoplasm. Gain-of-function and loss-of-function studies in CRC cell lines indicated that circVAPA could promote CRC cell proliferation, migration, invasion, and inhibit apoptosis. miRanda software (v3.3a) was used to predict target miRNAs of circVAPA. Moreover, target miRNAs associated with the KEGG pathway of COLORECTAL CANCER (Entry: map05210; https://www.kegg.jp/) were screened using DIANA-miRPath v.3 platform (Reverse Search module; TarBase v7.0 method). The analyses by miRanda and miRPath suggested that circVAPA could potentially bind to hsa-miR-101-3p (miR-101) associated with the COLORECTAL CANCER pathway. Luciferase reporter assay confirmed a direct interaction between circVAPA and miR-101. Furthermore, circVAPA had no effect on the expression level of miR-101, and miR-101 over-expression had the similar tumor-suppressing effects as circVAPA silencing. The tumor-promoting effect of circVAPA over-expression could be reversed by the up-regulation of miR-101. These data demonstrated that circVAPA promoted CRC progression by sponging miR-101. In conclusion, we have verified that circVAPA is up-regulated in CRC patients' tissues and plasma, and exerts oncogenic properties by sponging miR-101 in CRC. CircVAPA could serve as a promising biomarker and a therapeutic target for CRC.


Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias Colorretais/metabolismo , MicroRNAs/biossíntese , RNA/biossíntese , Regulação para Cima/fisiologia , Proteínas de Transporte Vesicular/biossíntese , Idoso , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , RNA/genética , Proteínas de Transporte Vesicular/genética
12.
Biomed Pharmacother ; 111: 596-601, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30611100

RESUMO

BACKGROUND: Increasing evidences demonstrate that circular RNAs (circRNAs) play an important role in the development and progression of human cancers. Nevertheless, the functions and molecular mechanism of circRNAs in esophageal squamous cell carcinoma (ESCC) tumorigenesis are largely unknown. The purpose of this research is to investigate the expression and potential role of a new circular RNA named circ-SMAD7 on ESCC carcinogenesis. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure circ-SMAD7 expression amount in both ESCC plasmas and tissues. Then the correlation between the expression of circ-SMAD7 and clinicopathological features was analyzed. Furthermore, by loss-of-function and gain-of-function experiments in ESCC cells, a functional analysis of circ-SMAD7 on ESCC cell proliferation and migration was performed. RESULTS: The expression of circ-SMAD7 was validated to be significantly down-regulated in ESCC plasmas in comparison with normal controls, showing a high negative correlation with TNM stage (P = 0.000) and lymphatic metastasis (P = 0.000). Moreover, circ-SMAD7 was significantly down-regulated in ESCC tissues compared to adjacent normal tissues. Furthermore, Loss-of-function and gain-of-function experiments revealed that the expression level of circ-SMAD7 affected the proliferation and migration of ESCC cell lines. CONCLUSION: Our study firstly exposed that over-expressioncirc-SMAD7, an influential regulator in cancer progression, can inhibit tumor proliferation and migration in ESCC and have the potential of becoming a biomarker for the diagnosis and therapy of ESCC.


Assuntos
Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , RNA/biossíntese , Proteína Smad7/biossíntese , Idoso , Linhagem Celular Tumoral , Neoplasias Esofágicas/prevenção & controle , Carcinoma de Células Escamosas do Esôfago/prevenção & controle , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regulação para Cima/fisiologia
13.
FEBS Lett ; 593(3): 361-368, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30536890

RESUMO

Bacterial RNA polymerase (RNAP) serves as a primase during replication of single-stranded plasmids and filamentous phages. Primer RNA (prRNA) synthesis from the origin regions of these replicons depends on the σ factor that normally participates in promoter recognition. However, it was proposed that σ may not be required for origin recognition but is rather involved in RNA extension by RNAP. Here, by analyzing the natural replication origin of bacteriophage M13 and synthetic ssDNA templates, we show that interactions of σ with promoter-like motifs stabilize priming complexes and can control prRNA synthesis by trapping RNAP on the template. Thus, the σ factor is involved in both DNA recognition and RNA priming, unifying its functions in transcription initiation from double- and single-stranded templates.


Assuntos
RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Regiões Promotoras Genéticas , RNA Bacteriano/biossíntese , RNA/biossíntese , Fator sigma/química , Bacteriófago M13/química , Bacteriófago M13/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA Viral/química , DNA Viral/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA/química , Fator sigma/metabolismo
14.
Nucleic Acids Res ; 47(4): 1725-1739, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30544263

RESUMO

The path from DNA to RNA to protein in eukaryotes is guided by a series of factors linking transcription, mRNA export and translation. Many of these are conserved from yeast to humans. Trypanosomatids, which diverged early in the eukaryotic lineage, exhibit unusual features such as polycistronic transcription and trans-splicing of all messenger RNAs. They possess basal transcription factors, but lack recognisable orthologues of many factors required for transcription elongation and mRNA export. We show that retrotransposon hotspot (RHS) proteins fulfil some of these functions and that their depletion globally impairs nascent RNA synthesis by RNA polymerase II. Three sub-families are part of a coordinated process in which RHS6 is most closely associated with chromatin, RHS4 is part of the Pol II complex and RHS2 connects transcription with the translation machinery. In summary, our results show that the components of eukaryotic transcription are far from being universal, and reveal unsuspected plasticity in the course of evolution.


Assuntos
Proteínas de Protozoários/genética , RNA/biossíntese , Retroelementos/genética , Transcrição Genética , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/genética , Cromatina/genética , DNA de Protozoário/genética , Eucariotos/genética , Variação Genética/genética , Humanos , Regiões Promotoras Genéticas/genética , RNA/genética , RNA Polimerase II/genética , Trypanosoma brucei brucei/genética
15.
Biomed Pharmacother ; 111: 386-394, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30594051

RESUMO

BACKGROUND: Treg/Th17 imbalance plays an essential role in the pathogenesis of asthma. Disordered LncRNAs were observed in asthma, however, whether LncRNAs can regulate the Treg/Th17 balance and its mechanism still needs to be investigated. METHODS: Microarrays were performed to identify the differentially expressed lncRNAs and microRNAs in peripheral blood CD4 + T cells from patients with asthma and healthy controls. Bioinformatical evidence was used to select candidate lncRNAs and microRNAs which may involve in regulation of Treg/Th17 balance. The function of LncRNA-MEG3 and microRNA-17 on the alteration of the CD4 + T cell population were determined in vitro experiments. Meanwhile, the regulatory effect of LncRNA-MEG3 and microRNA-17 on RORγt or Foxp3 was estimated. The interaction of LncRNA-MEG3 with microRNA-17 was confirmed by dual luciferase reporter assay and RNA pull-down. RESULTS: 25 lncRNAs and 19 microRNAs were selected as candidate genes which differentially expressed in CD4 + T cells from patients with asthma compared with healthy controls and had potential to control Treg/Th17 balance by regulating RORγt or Foxp3. Alternation of LncRNA-MEG3 changed the function and increased the percentage of Th17. LncRNA-MEG3 could regulate the RORγt mRNA and protein level. LncRNA-MEG3 could inhibit the level of microRNA-17 as a competing endogenous RNA (ceRNA). microRNA-17 suppressed Th17 though targeting RORγt directly. CONCLUSION: LncRNA-MEG3 can sponge microRNA-17 as a ceRNA, thereby regulating RORγt and ultimately affecting Treg/Th17 balance in asthma. The lncRNA/microRNA axis may have potential application in clinical treatment and diagnosis of the disease.


Assuntos
Asma/metabolismo , MicroRNAs/biossíntese , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/biossíntese , RNA Longo não Codificante/biossíntese , Linfócitos T Reguladores/fisiologia , Células Th17/fisiologia , Adulto , Asma/genética , Feminino , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , RNA/biossíntese , RNA/genética , RNA Longo não Codificante/genética
16.
Mol Cell ; 73(3): 519-532.e4, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30554946

RESUMO

Transcriptional regulation occurs via changes to rates of different biochemical steps of transcription, but it remains unclear which rates are subject to change upon biological perturbation. Biochemical studies have suggested that stimuli predominantly affect the rates of RNA polymerase II (Pol II) recruitment and polymerase release from promoter-proximal pausing. Single-cell studies revealed that transcription occurs in discontinuous bursts, suggesting that features of such bursts like frequency and intensity could also be regulated. We combined Pol II chromatin immunoprecipitation sequencing (ChIP-seq) and single-cell transcriptional measurements to show that an independently regulated burst initiation step is required before polymerase recruitment can occur. Using a number of global and targeted transcriptional regulatory perturbations, we showed that biological perturbations regulated both burst initiation and polymerase pause release rates but seemed not to regulate polymerase recruitment rate. Our results suggest that transcriptional regulation primarily acts by changing the rates of burst initiation and polymerase pause release.


Assuntos
Células-Tronco Embrionárias Murinas/enzimologia , RNA Polimerase II/metabolismo , RNA/biossíntese , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Ativação Transcricional , Animais , Sítios de Ligação , Linhagem Celular , Simulação por Computador , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Modelos Genéticos , Ligação Proteica , RNA/genética , RNA Polimerase II/genética , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Fatores de Tempo
17.
Biomed Pharmacother ; 109: 1709-1717, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30551425

RESUMO

BACKGROUND: Circular RNAs (circRNAs) comprise a novel class of noncoding RNAs that play important roles in a variety of diseases. However, the mechanism by which circRNAs regulate the osteogenic differentiation of maxillary sinus membrane stem cells (MSMSCs) remains largely unclear. METHODS: Microarray analysis was used to explore the expression profiles of circRNAs during the osteogenic differentiation of normal and BMP2 induced-MSMSCs. CircRNA_33287 was identified by agarose electrophoresis, quantitative real-time PCR (qRT-PCR), and western blotting. The function of circRNA_33287 was assessed by loss- and gain-of-function techniques and Alizarin red staining. Potential miRNA binding sites for circRNA_33287, and the target genes of miR-214-3p, were predicted by using online bioinformatics analysis tools. The relationships among the regulatory roles played by circRNA_33287, miR-214-3p, and Runt-related transcription factor 3 (Runx3), during the osteogenic differentiation of MSMSCs were verified by use of the dual luciferase reporter assay, qRT-PCR, and western blotting techniques, respectively. In addition, the molecular sponge potential of circRNA_33287 for miRNA was assessed via in vivo ectopic bone formation and a histological analysis performed after hematoxylin and eosin staining. RESULTS: Expression of circRNA_33287 was confirmed to be up-regulated during the osteogenic differentiation of MSMSCS. Overexpression and silencing of circRNA_33287 increased and decreased the expression levels of key markers of osteogenesis, respectively, including Runx2, OSX, and ALP. Furthermore, circRNA_33287 acted as a molecular sponge for miR-214-3p, which regulated Runx3 expression by targeting its 3'UTR. Moreover, circRNA_33287 protected Runx3 from miR-214-3p-mediated suppression. In addition, circRNA_33287 was shown to increase ectopic bone formation in vivo and displayed the strongest ability to stimulate bone formation when co-transfected with a miR-214-3p inhibitor. CONCLUSION: The novel pathway circRNA_33287/miR-214-3p/Runx3 was found to play a role in regulating the osteoblastic differentiation of MSMSCs in the posterior maxilla.


Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/biossíntese , Seio Maxilar/metabolismo , MicroRNAs/biossíntese , Osteogênese/fisiologia , RNA/biossíntese , Células-Tronco/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Células HEK293 , Humanos , Masculino , Seio Maxilar/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Longo não Codificante/biossíntese
18.
PLoS One ; 13(12): e0208947, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30532129

RESUMO

Purines are essential molecules for all forms of life. In addition to constituting a backbone of DNA and RNA, purines play roles in many metabolic pathways, such as energy utilization, regulation of enzyme activity, and cell signaling. The supply of purines is provided by two pathways: the salvage pathway and de novo synthesis. Although purine de novo synthesis (PDNS) activity varies during the cell cycle, this pathway represents an important source of purines, especially for rapidly dividing cells. A method for the detailed study of PDNS is lacking for analytical reasons (sensitivity) and because of the commercial unavailability of the compounds. The aim was to fully describe the mass spectrometric fragmentation behavior of newly synthesized PDNS-related metabolites and develop an analytical method. Except for four initial ribotide PDNS intermediates that preferentially lost water or phosphate or cleaved the forming base of the purine ring, all the other metabolites studied cleaved the glycosidic bond in the first fragmentation stage. Fragmentation was possible in the third to sixth stages. A liquid chromatography-high-resolution mass spectrometric method was developed and applied in the analysis of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual enzymatic steps of PDNS and the salvage pathway. The identities of the newly synthesized intermediates of PDNS were confirmed by comparing the fragmentation patterns of the synthesized metabolites with those produced by cells (formed under pathological conditions of known and theoretically possible defects of PDNS). The use of stable isotope incorporation allowed the confirmation of fragmentation mechanisms and provided data for future fluxomic experiments. This method may find uses in the diagnosis of PDNS disorders, the investigation of purinosome formation, cancer research, enzyme inhibition studies, and other applications.


Assuntos
DNA/biossíntese , Purinas/biossíntese , RNA/biossíntese , Espectrometria de Massas em Tandem , Sistemas CRISPR-Cas , Cromatografia Líquida , DNA/química , Edição de Genes , Células HeLa , Humanos , Purinas/química , RNA/química
19.
J Exp Clin Cancer Res ; 37(1): 325, 2018 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-30591054

RESUMO

BACKGROUND: Circular RNA (circRNA) is a novel class of noncoding RNAs with functions in various pathophysiological activities. However, the expression profiles and functions of circRNAs in colorectal cancer (CRC) remain largely unknown. METHODS: High-throughput RNA sequencing (RNA-seq) was performed to assess circRNA expression profiles in 4 paired CRC tissues, and significantly dysregulated circRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to predict the potential functions of dysregulated circRNAs. Target miRNAs of circRNAs were predicted using miRanda software, and were further analyzed combining DIANA-miRPath v.3 platform (Reverse Search module) with KEGG pathways of COLORECTAL CANCER and MicroRNAs in cancer (Entry: map05210 and map05206). CircRNA-miRNA interaction networks were constructed using Cytoscape software. Expression levels of a significantly down-regulated circRNA, circDDX17 (hsa_circ_0002211), was detected by qRT-PCR in 60 paired CRC tissues. CircDDX17 was knockdown by siRNA, and the biological functions of circDDX17 were examined in CRC cell lines. RESULTS: Totally 448 differentially expressed circRNAs were identified, including 394 up-regulated and 54 down-regulated circRNAs. qRT-PCR validation confirmed the reliability of the RNA-Seq data. GO and KEGG analyses revealed that these dysregulated circRNAs were potentially implicated in CRC pathogenesis. Analyses by combining miRanda and miRPath softwares with KEGG pathways suggested that the miRNAs targeted by the top 10 dysregulated circRNAs were associated with the KEGG pathways of COLORECTAL CANCER and MicroRNAs in cancer, indicating that circRNA-miRNA interactions might play important functional roles in the initiation and progression of CRC. The results of qRT-PCR for circDDX17 in 60 paired CRC tissues showed that circDDX17 was significantly down-regulated in CRC tissues and associated with unfavorable clinicopathological parameters. In vitro experiments showed that silencing of circDDX17 promoted CRC cell proliferation, migration, invasion, and inhibited apoptosis. CONCLUSIONS: In conclusion, we have identified numerous circRNAs that are dysregulated in CRC tissues compared with adjacent normal mucosa tissues. Bioinformatic analyses suggested that these dysregulated circRNAs might play important functional roles in CRC tumorigenesis. CircDDX17 functions as a tumor suppressor and could serve as a potential biomarker and a therapeutic target for CRC.


Assuntos
Neoplasias Colorretais/genética , RNA Helicases DEAD-box/genética , RNA/genética , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Biologia Computacional/métodos , Genes Supressores de Tumor , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA/biossíntese , RNA/metabolismo , Análise de Sequência de RNA/métodos , Transfecção
20.
Proc Natl Acad Sci U S A ; 115(28): E6437-E6446, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29946020

RESUMO

Single-cell RNA sequencing (scRNA-seq) enables the quantification of each gene's expression distribution across cells, thus allowing the assessment of the dispersion, nonzero fraction, and other aspects of its distribution beyond the mean. These statistical characterizations of the gene expression distribution are critical for understanding expression variation and for selecting marker genes for population heterogeneity. However, scRNA-seq data are noisy, with each cell typically sequenced at low coverage, thus making it difficult to infer properties of the gene expression distribution from raw counts. Based on a reexamination of nine public datasets, we propose a simple technical noise model for scRNA-seq data with unique molecular identifiers (UMI). We develop deconvolution of single-cell expression distribution (DESCEND), a method that deconvolves the true cross-cell gene expression distribution from observed scRNA-seq counts, leading to improved estimates of properties of the distribution such as dispersion and nonzero fraction. DESCEND can adjust for cell-level covariates such as cell size, cell cycle, and batch effects. DESCEND's noise model and estimation accuracy are further evaluated through comparisons to RNA FISH data, through data splitting and simulations and through its effectiveness in removing known batch effects. We demonstrate how DESCEND can clarify and improve downstream analyses such as finding differentially expressed genes, identifying cell types, and selecting differentiation markers.


Assuntos
Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Genéticos , RNA/biossíntese , RNA/genética , Animais , Humanos
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