Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 15.595
Filtrar
1.
Nucleic Acids Res ; 48(14): 7712-7727, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32805052

RESUMO

Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.


Assuntos
Quinases Ciclina-Dependentes/fisiologia , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Cromatina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Células HEK293 , Humanos , Mutação , Fosforilação , RNA/biossíntese , RNA Polimerase II/química , Análise de Sequência de RNA , Serina/metabolismo , Fatores de Elongação da Transcrição/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(35): 21274-21280, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817521

RESUMO

We report here crystal structures of a reverse transcriptase RTX, which was evolved in vitro from the B family polymerase KOD, in complex with either a DNA duplex or an RNA-DNA hybrid. Compared with the apo, binary, and ternary complex structures of the original KOD polymerase, the 16 substitutions that result in the function of copying RNA to DNA do not change the overall protein structure. Only six substitutions occur at the substrate-binding surface, and the others change domain-domain interfaces in the polymerase to enable RNA-DNA hybrid binding and reverse transcription. Most notably, F587L at the Palm and Thumb interface stabilizes the open and apo conformation of the Thumb. The intrinsically flexible Thumb domain seems to play a major role in accommodating the RNA-DNA hybrid product distal to the active site. This is reminiscent of naturally occurring RNA-dependent DNA polymerases, including telomerase, which have a dramatically augmented Thumb domain, and of reverse transcriptase, which extends its Thumb with the RNase H domain.


Assuntos
Evolução Molecular , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , RNA/biossíntese , Substituição de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Conformação Proteica , DNA Polimerase Dirigida por RNA/química
3.
BMC Evol Biol ; 20(1): 75, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32590933

RESUMO

BACKGROUND: We hypothesize prebiotic evolution of self-replicating macro-molecules (Alberts, Molecular biology of the cell, 2015; Orgel, Crit Rev Biochem Mol Biol 39:99-123, 2004; Hud, Nat Commun 9:5171) favoured the constituent nucleotides and biophysical properties observed in the RNA and DNA of modern organisms. Assumed initial conditions are a shallow tide pool, containing a racemic mix of diverse nucleotide monomers (Barks et al., Chembiochem 11:1240-1243, 2010; Krishnamurthy, Nat Commun 9:5175, 2018; Hirao, Curr Opin Chem Biol 10:622-627), subject to day/night thermal fluctuations (Piccirilli et al., Nature 343:33-37, 1990). Self-replication, like Polymerase Chain Reactions, followed as higher daytime thermal energy "melted" inter-strand hydrogen bonds causing strand separation while solar UV radiation increased prebiotic nucleobase formation (Szathmary, Proc Biol Sci 245:91-99, 1991; Materese et al., Astrobiology 17:761-770, 2017; Bera et al., Astrobiology 17:771-785, 2017). Lower night energies allowed free monomers to form hydrogen bonds with their template counterparts leading to daughter strand synthesis (Hirao, Biotechniques 40:711, 2006). RESULTS: Evolutionary selection favoured increasing strand length to maximize auto-catalytic function in RNA and polymer stability in double stranded DNA (Krishnamurthy, Chemistry 24:16708-16715, 2018; Szathmary, Nat Rev Genet 4:995-1001, 2003). However, synthesis of the full daughter strand before daytime temperatures produced strand separation, longer polymer length required increased speed of self-replication. Computer simulations demonstrate optimal polynucleotide autocatalytic speed is achieved when the constituent nucleotides possess a left-right asymmetry that decreases the hydrogen bond kinetic barrier for the free nucleotide attachment to the template on one side and increases bond barrier on the other side preventing it from releasing prior to covalent bond formation. This phenomenon is similar to asymmetric kinetics observed during polymerization of the front and the back ends of linear cytoskeletal proteins such as actin and microtubules (Orgel, Nature 343:18-20, 1990; Henry, Curr Opin Chem Biol 7:727-733, 2003; Walker et al., J Cell Biol 108:931-937, 1989; Crevenna et al., J Biol Chem 288:12102-12113, 2013). Since rotation of the nucleotide would disrupt the asymmetry, the optimal nucleotides must form two or more hydrogen bonds with their counterpart on the template strand. All nucleotides in modern RNA and DNA have these predicted properties. Our models demonstrate these constraints on the properties of constituent monomers result in biophysical properties found in modern DNA and RNA including strand directionality, anti-parallel strand orientation, homochirality, quadruplet alphabet, and complementary base pairing. Furthermore, competition between RNA and DNA auto-replicators for 3 nucleotides in common permit states coexistence and possible cooperative interactions that could be incorporated into nascent living systems. CONCLUSION: Our findings demonstrate the molecular properties of DNA/RNA could have emerged from Darwinian competition among macromolecular replicators that selected nucleotide monomers that maximized the speed of autocatalysis.


Assuntos
Replicação do DNA , DNA/biossíntese , Polinucleotídeos/biossíntese , Prebióticos , RNA/biossíntese , DNA/genética , Cinética , Polinucleotídeos/genética , RNA/genética
4.
J Hematol Oncol ; 13(1): 43, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: covidwho-165275

RESUMO

The novel coronavirus (2019-nCoV) is an emerging causative agent that was first described in late December 2019 and causes a severe respiratory infection in humans. Notably, many of affected patients of COVID-19 were people with malignancies. Moreover, cancer has been identified as an individual risk factor for COVID-19. In addition, the expression of angiotensin converting enzyme 2 (ACE2), the receptor of COVID-19, were aberrantly expressed in many tumors. However, a systematic analysis of ACE2 aberration remained to be elucidated in human cancers. Here, we analyzed genetic alteration, RNA expression, and DNA methylation of ACE2 across over 30 tumors. Notably, overexpression of ACE2 have been observed in including colon adenocarcinoma (COAD), kidney renal papillary cell carcinoma (KIRP), pancreatic adenocarcinoma (PAAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), and lung adenocarcinoma (LUAD). In addition, hypo DNA methylation of ACE2 has also been identified in most of these ACE2 highly expressed tumors. Conclusively, our study for the first time curated both genetic and epigenetic variations of ACE2 in human malignancies. Notably, because our study is a bioinformatics assay, further functional and clinical validation is warranted.


Assuntos
Betacoronavirus , Infecções por Coronavirus , Neoplasias/enzimologia , Pandemias , Peptidil Dipeptidase A , Pneumonia Viral , Receptores Virais , Infecções por Coronavirus/enzimologia , Infecções por Coronavirus/etiologia , Infecções por Coronavirus/virologia , Metilação de DNA , DNA de Neoplasias/metabolismo , Humanos , Neoplasias/complicações , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/genética , Pneumonia Viral/enzimologia , Pneumonia Viral/etiologia , Pneumonia Viral/virologia , RNA/biossíntese , Receptores Virais/biossíntese , Receptores Virais/genética
5.
J Hematol Oncol ; 13(1): 43, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366279

RESUMO

The novel coronavirus (2019-nCoV) is an emerging causative agent that was first described in late December 2019 and causes a severe respiratory infection in humans. Notably, many of affected patients of COVID-19 were people with malignancies. Moreover, cancer has been identified as an individual risk factor for COVID-19. In addition, the expression of angiotensin converting enzyme 2 (ACE2), the receptor of COVID-19, were aberrantly expressed in many tumors. However, a systematic analysis of ACE2 aberration remained to be elucidated in human cancers. Here, we analyzed genetic alteration, RNA expression, and DNA methylation of ACE2 across over 30 tumors. Notably, overexpression of ACE2 have been observed in including colon adenocarcinoma (COAD), kidney renal papillary cell carcinoma (KIRP), pancreatic adenocarcinoma (PAAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), and lung adenocarcinoma (LUAD). In addition, hypo DNA methylation of ACE2 has also been identified in most of these ACE2 highly expressed tumors. Conclusively, our study for the first time curated both genetic and epigenetic variations of ACE2 in human malignancies. Notably, because our study is a bioinformatics assay, further functional and clinical validation is warranted.


Assuntos
Betacoronavirus , Infecções por Coronavirus , Neoplasias/enzimologia , Pandemias , Peptidil Dipeptidase A , Pneumonia Viral , Receptores Virais , Infecções por Coronavirus/enzimologia , Infecções por Coronavirus/etiologia , Infecções por Coronavirus/virologia , Metilação de DNA , DNA de Neoplasias/metabolismo , Humanos , Neoplasias/complicações , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/genética , Pneumonia Viral/enzimologia , Pneumonia Viral/etiologia , Pneumonia Viral/virologia , RNA/biossíntese , Receptores Virais/biossíntese , Receptores Virais/genética
6.
Am J Physiol Regul Integr Comp Physiol ; 319(1): R50-R58, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32432913

RESUMO

The current study explored whether the marked hypertrophic response noted with a short-term unilateral concurrent exercise paradigm was associated with more prominent changes in myonuclei accretion, ribosome biogenesis, and capillarization compared with resistance exercise alone (RE). Ten men (age 25 ± 4 yr) performed aerobic and resistance exercise (AE + RE) for one leg while the other leg did RE. Muscle biopsies were obtained before and after 5 wk of training and subjected to fiber-type specific immunohistochemical analysis, and quantification of total RNA content and mRNA/rRNA transcript abundance. Type II fiber cross-sectional area (CSA) increased with both AE + RE (22%) and RE (16%), while type I fiber CSA increased mainly with AE + RE (16%). The change score tended to differ between legs for type I CSA (P = 0.099), and the increase in smallest fiber diameter was greater in AE + RE than RE (P = 0.029). The number of nuclei per fiber increased after AE + RE in both fiber types, and this increase was greater (P = 0.027) than after RE. A strong correlation was observed between changes in number of nuclei per fiber and fiber CSA in both fiber types, for both AE + RE and RE (r > 0.8, P < 0.004). RNA content increased after AE + RE (24%, P = 0.019), but the change-scores did not differ across legs. The capillary variables generally increased in both fiber types, with no difference across legs. In conclusion, the accentuated hypertrophic response to AE + RE was associated with more pronounced myonuclear accretion, which was strongly correlated with the degree of fiber hypertrophy. This suggests that myonuclear accretion could play a role in facilitating muscle hypertrophy also during very short training periods.


Assuntos
Núcleo Celular/metabolismo , Exercício Físico/fisiologia , Músculo Esquelético/fisiologia , Adulto , Capilares/fisiologia , Humanos , Hipertrofia , Perna (Membro)/anatomia & histologia , Perna (Membro)/fisiologia , Imagem por Ressonância Magnética , Masculino , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares de Contração Lenta/fisiologia , Fibras Musculares de Contração Lenta/ultraestrutura , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/ultraestrutura , Resistência Física , RNA/biossíntese , Treinamento de Resistência , Ribossomos/metabolismo , Adulto Jovem
7.
Neurochirurgie ; 66(3): 168-173, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32201238

RESUMO

PURPOSE: An accurate understanding of cellular biochemical changes in human intervertebral disc (IVD)s and the corresponding mechanisms during the developmental process still remain unknown and important for investigating the function of critical factors in normal IVD development as well as ascertaining the therapeutic targets for the IVD degeneration. METHODS: Under ethical conditions, human fetal cervical IVDs at 4, 5, and 6 months of pregnancy were collected at abortion surgery. Normal adult human C3-C7 cervical IVDs were taken from cadaveric donors. Sox9, Pax1, TGF-ß1 and type I/II collagen protein and RNA were detected. The number of positive cells was counted to calculate the optical density value for each factor. RESULTS: Sox9, Pax1, and TGF-ß1 expression in the IVD was remarkably reduced with the developmental stage. The location of high expression of Sox9, Pax1, and TGF-ß1 changed with the developmental stage, and migrated from the nucleus pulposus to the annulus fibrosus and endplate. Higher Sox9, Pax1, and TGF-ß1 expression was finally observed around the sclerotome of the vertebral body. The anabolism of type I/II collagens is significantly increased in the IVD in the mid-trimester fetus. CONCLUSIONS: Sox9, Pax1 and TGF-ß1 participate in the developmental process of the human IVD and vertebral body. However, these factors show a separate expression of mRNA and protein, suggesting that they are expressed in the strict time and spatial order.


Assuntos
Colágeno Tipo II/biossíntese , Colágeno Tipo I/biossíntese , Disco Intervertebral/crescimento & desenvolvimento , Disco Intervertebral/metabolismo , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição SOX9/biossíntese , Fator de Crescimento Transformador beta1/biossíntese , Adulto , Cadáver , Colágeno Tipo I/genética , Colágeno Tipo II/genética , Feminino , Humanos , Imuno-Histoquímica , Disco Intervertebral/embriologia , Degeneração do Disco Intervertebral , Fatores de Transcrição Box Pareados/genética , Gravidez , Segundo Trimestre da Gravidez , RNA/biossíntese , RNA/genética , Fatores de Transcrição SOX9/genética , Fator de Crescimento Transformador beta1/genética
8.
Science ; 368(6487)2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32217750

RESUMO

Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as the information carrier. Two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rolling-circle synthesis), and describe emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could be propagated as hereditary material in cellular contexts.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , RNA/biossíntese , Replicon , Transcrição Genética , Proteínas Virais/metabolismo , Biocatálise
9.
J Neurosci ; 40(15): 3130-3140, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32144181

RESUMO

Acoustic overexposure, such as listening to loud music too often, results in noise-induced hearing loss. The pathologies of this prevalent sensory disorder begin within the ear at synapses of the primary auditory receptors, their postsynaptic partners and their supporting cells. The extent of noise-induced damage, however, is determined by overstimulation of primary auditory receptors, upstream of where the pathologies manifest. A systematic characterization of the electrophysiological function of the upstream primary auditory receptors is warranted to understand how noise exposure impacts on downstream targets, where the pathologies of hearing loss begin. Here, we used the experimentally-accessible locust ear (male, Schistocerca gregaria) to characterize a decrease in the auditory receptor's ability to respond to sound after noise exposure. Surprisingly, after noise exposure, the electrophysiological properties of the auditory receptors remain unchanged, despite a decrease in the ability to transduce sound. This auditory deficit stems from changes in a specialized receptor lymph that bathes the auditory receptors, revealing striking parallels with the mammalian auditory system.SIGNIFICANCE STATEMENT Noise exposure is the largest preventable cause of hearing loss. It is the auditory receptors that bear the initial brunt of excessive acoustic stimulation, because they must convert excessive sound-induced movements into electrical signals, but remain functional afterward. Here we use the accessible ear of an invertebrate to, for the first time in any animal, characterize changes in auditory receptors after noise overexposure. We find that their decreased ability to transduce sound into electrical signals is, most probably, due to changes in supporting (scolopale) cells that maintain the ionic composition of the ear. An emerging doctrine in hearing research is that vertebrate primary auditory receptors are surprisingly robust, something that we show rings true for invertebrate ears too.


Assuntos
Gafanhotos , Perda Auditiva Provocada por Ruído/fisiopatologia , Membrana Timpânica/fisiopatologia , Animais , Vias Auditivas/fisiopatologia , Fenômenos Biomecânicos , Nervo Coclear/fisiopatologia , Fenômenos Eletrofisiológicos , Potenciais Evocados Auditivos , Potenciais Evocados Auditivos do Tronco Encefálico , Perda Auditiva Provocada por Ruído/genética , Linfa , Masculino , Mecanotransdução Celular , Ruído , RNA/biossíntese , RNA/genética
10.
Nat Commun ; 11(1): 30, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31937763

RESUMO

Systems biology has long been interested in models capturing both metabolism and expression in a cell. We propose here an implementation of the metabolism and expression model formalism (ME-models), which we call ETFL, for Expression and Thermodynamics Flux models. ETFL is a hierarchical model formulation, from metabolism to RNA synthesis, that allows simulating thermodynamics-compliant intracellular fluxes as well as enzyme and mRNA concentration levels. ETFL formulates a mixed-integer linear problem (MILP) that enables both relative and absolute metabolite, protein, and mRNA concentration integration. ETFL is compatible with standard MILP solvers and does not require a non-linear solver, unlike the previous state of the art. It also accounts for growth-dependent parameters, such as relative protein or mRNA content. We present ETFL along with its validation using results obtained from a well-characterized E. coli model. We show that ETFL is able to reproduce proteome-limited growth. We also subject it to several analyses, including the prediction of feasible mRNA and enzyme concentrations and gene essentiality.


Assuntos
Modelos Biológicos , Biologia de Sistemas/métodos , Termodinâmica , Biomassa , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Engenharia Metabólica , Metabolômica , Proteoma/metabolismo , Proteômica , RNA/biossíntese , RNA Mensageiro/metabolismo , Software
11.
RNA ; 26(3): 345-360, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900329

RESUMO

The use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. The technology used for the synthesis of these in vitro-transcribed RNAs, predominantly using phage RNA polymerases (RNAPs), is well established. However, transcripts synthesized with RNAPs are known to display an immune-stimulatory activity in vivo that is often undesirable. Previous studies have identified double-stranded RNA (dsRNA), a major by-product of the in vitro transcription (IVT) process, as a trigger of cellular immune responses. Here we describe the characterization of a high-temperature IVT process using thermostable T7 RNAPs to synthesize functional mRNAs that demonstrate reduced immunogenicity without the need for a post-synthesis purification step. We identify features that drive the production of two kinds of dsRNA by-products-one arising from 3' extension of the run-off product and one formed by the production of antisense RNAs-and demonstrate that at a high temperature, T7 RNAP has reduced 3'-extension of the run-off product. We show that template-encoded poly(A) tailing does not affect 3'-extension but reduces the formation of the antisense RNA by-products. Combining high-temperature IVT with template-encoded poly(A) tailing prevents the formation of both kinds of dsRNA by-products generating functional mRNAs with reduced immunogenicity.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Antissenso/biossíntese , RNA de Cadeia Dupla/genética , RNA/genética , Bacteriófago T7/enzimologia , Bacteriófago T7/genética , Imunidade Celular/genética , RNA/biossíntese , RNA Antissenso/genética , RNA Mensageiro/genética , Transcrição Genética
12.
Toxicol In Vitro ; 62: 104686, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31614173

RESUMO

Cadmium exposure has raised great public concern. Extensive studies have revealed the neurotoxic effects of cadmium exposure during brain development. However, more evidence is still needed to reach a consistent conclusion and uncover the underlying mechanisms. Here, we used primary mouse embryonic neural stem/progenitor cells (NSPCs) as a cell model and exposed the cells to 0, 1, 2 or 4 µM cadmium. High-throughput mRNA-seq technology was used to explore the global transcriptome changes in NSPCs after exposure to 2 µM cadmium. We found that cadmium exposure remarkably influenced the expression of genes involved in cell growth, proliferation, cell cycle and survival. Pathway-Act-Network analysis revealed that these altered genes were targeted to the P53, PI3K-AKT, MAPK, calcium, and NF-kappa B signaling pathways. In vitro experiments using cultured NSPCs verified that cadmium exposure reduced cell viability, proliferation, neurosphere formation and caused cell cycle arrest at low concentrations (≤ 2 µM), while induced cell apoptosis at high concentrations (≥ 4 µM). Real-time PCR results confirmed the concentration-dependent effects of cadmium exposure on the expression of critical genes in the above signaling pathways. Together, our results provide transcriptomic insight into cadmium-induced developmental neurotoxic effects and the underlying mechanisms.


Assuntos
Cádmio/toxicidade , Células-Tronco Neurais/efeitos dos fármacos , Síndromes Neurotóxicas/patologia , Transcriptoma/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos BALB C , Neurogênese/efeitos dos fármacos , Gravidez , RNA/biossíntese , RNA/genética , Transdução de Sinais/efeitos dos fármacos
13.
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 Circular , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Regulação para Cima
14.
Inflamm Res ; 68(12): 1025-1034, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31531682

RESUMO

OBJECTIVE: Saikosaponin c (SSc), a compound purified from the traditional Chinese herb of Radix Bupleuri was previously identified to exhibit anti-HBV replication activity. However, the mechanism through which SSc acts against HBV remains unknown. In this study, we investigated the mechanism of SSc mediated anti-HBV activity. METHODS: HepG2.2.15 cells were cultured at 37 â„ƒ in the presence of 1-40 µg/mL of SSc or DMSO as a control. The expression profile of HBV markers, cytokines, HNF1α and HNF4α were investigated by real-time quantitative PCR, Elisa, Western blot and Dot blotting. Knockdown of HNF1α or HNF4α in HepG2.2.15 cells was mediated by two small siRNAs specifically targeting HNF1α or HNF4α. RESULTS: We found that SSc stimulates IL-6 expression, leading to attenuated HNF1α and HNF4α expression, which further mediates suppression of HBV pgRNA synthesis. Knockdown of HNF1α or HNF4α in HepG2.2.15 cells by RNA interference abrogates SSc's anti-HBV role. Moreover, SSc is effective to both wild-type and drug-resistant HBV mutants. CONCLUSION: SSc inhibits pgRNA synthesis by targeting HNF1α and HNF4α. These results indicate that SSc acts as a promising compound for modulating pgRNA transcription in the therapeutic strategies against HBV infection.


Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Ácido Oleanólico/análogos & derivados , RNA Viral/biossíntese , RNA/biossíntese , Saponinas/farmacologia , Células Hep G2 , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Humanos , Ácido Oleanólico/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética
15.
Mol Cell ; 75(6): 1161-1177.e11, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31421980

RESUMO

Genes are transcribed in a discontinuous pattern referred to as RNA bursting, but the mechanisms regulating this process are unclear. Although many physiological signals, including glucocorticoid hormones, are pulsatile, the effects of transient stimulation on bursting are unknown. Here we characterize RNA synthesis from single-copy glucocorticoid receptor (GR)-regulated transcription sites (TSs) under pulsed (ultradian) and constant hormone stimulation. In contrast to constant stimulation, pulsed stimulation induces restricted bursting centered around the hormonal pulse. Moreover, we demonstrate that transcription factor (TF) nuclear mobility determines burst duration, whereas its bound fraction determines burst frequency. Using 3D tracking of TSs, we directly correlate TF binding and RNA synthesis at a specific promoter. Finally, we uncover a striking co-bursting pattern between TSs located at proximal and distal positions in the nucleus. Together, our data reveal a dynamic interplay between TF mobility and RNA bursting that is responsive to stimuli strength, type, modality, and duration.


Assuntos
Glucocorticoides/farmacologia , Regiões Promotoras Genéticas , RNA/biossíntese , Receptores de Glucocorticoides/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Genética/efeitos dos fármacos , Animais , Camundongos , RNA/genética
16.
Nucleic Acids Res ; 47(19): e118, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31392994

RESUMO

In vitro synthesized RNA is used widely in studies of RNA biology, biotechnology and RNA therapeutics. However, in vitro synthesized RNA often contains impurities, such as RNAs with lengths shorter and longer than the expected runoff RNA. We have recently confirmed that longer RNA products are formed predominantly via cis self-primed extension, in which released runoff RNA folds back on itself to prime its own RNA-templated extension. In the current work, we demonstrate that addition of a DNA oligonucleotide (capture DNA) that is complementary to the 3' end of the expected runoff RNA effectively prevents self-primed extension, even under conditions commonly used for high RNA yields. Moreover, the presence of this competing capture DNA during 'high yield' transcription, leads to an increase in the yield of expected runoff RNA by suppressing the formation of undesired longer RNA byproducts.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , DNA/genética , RNA/biossíntese , Transcrição Genética , Proteínas Virais/genética , Bacteriófago T7/genética , Sequência de Bases/genética , RNA Polimerases Dirigidas por DNA/química , Cinética , Oligonucleotídeos/genética , RNA/genética , Dobramento de RNA/genética , Moldes Genéticos , Proteínas Virais/química
17.
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
18.
Histochem Cell Biol ; 152(4): 271-280, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31346697

RESUMO

In human cells, the intergenic spacers (IGS), which separate ribosomal genes, are complex approximately 30 kb-long loci. Recent studies indicate that all, or almost all, parts of IGS may be transcribed, and that at least some of them are involved in the regulation of the ribosomal DNA (rDNA) transcription, maintenance of the nucleolar architecture, and response of the cell nucleus to stress. However, since each cell contains hundreds not quite identical copies of IGS, the structure and functions of this locus remain poorly understood, and the dynamics of its products has not been specially studied. In this work, we used quantitative PCR to measure the expression levels of various rDNA regions at different times after inhibition of the transcription by Actinomycin D applied in high doses. This approach allowed us to measure real or extrapolated half-life times of some IGS loci. Our study reveals characteristic dynamic patterns suggestive of various pathways of RNA utilization and decay.


Assuntos
DNA Espaçador Ribossômico/metabolismo , RNA/biossíntese , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Células HeLa , Humanos , RNA/análise , RNA/genética , RNA/isolamento & purificação
19.
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
20.
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 Circular , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA