RESUMEN

We previously showed that stroke alters circular RNA (circRNA) expression profiles. Many circRNAs undergo epitranscriptomic modifications, particularly methylation of adenosine to form N6-methyladenosine (m6A). This modification significantly influences the circRNA metabolism and functionality. Hence, we currently evaluated if transient focal ischemia in adult C57BL/6J mice alters the m6A methylation of circRNAs. Changes in m6A were profiled in the peri-infarct cortex following immunoprecipitation coupled with microarrays. Correlation and gene ontology analyses were performed to understand the association of m6A changes with circRNA regulation and functional implications after stroke. Many circRNAs showed differential regulation (up or down) after stroke, and this change was highest at 24h of reperfusion. Notably, most circRNAs differentially regulated after stroke also exhibited temporal changes in m6A modification patterns. The majority of circRNAs that showed post-stroke differential m6A modifications were derived from protein-coding genes. Hyper-than hypomethylation of circRNAs was most prevalent after stroke. Gene ontology analysis of the host genes suggested that m6A-modified circRNAs might regulate functions such as synapse-related processes, indicating that m6A epitranscriptomic modification in circRNAs could potentially influence post-stroke synaptic pathophysiology.

Asunto(s)

Adenosina , Ratones Endogámicos C57BL , ARN Circular , Accidente Cerebrovascular , Animales , ARN Circular/genética , ARN Circular/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Masculino , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Ratones , ARN/genética , ARN/biosíntesis , Metilación

RESUMEN

Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers1,2. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations3. However, fundamental questions about the temporal regulation of transcription and enhancer-gene coordination remain unanswered, primarily because of the absence of a single-cell perspective on active transcription. In this study, we present scGRO-seq-a new single-cell nascent RNA sequencing assay that uses click chemistry-and unveil coordinated transcription throughout the genome. We demonstrate the episodic nature of transcription and the co-transcription of functionally related genes. scGRO-seq can estimate burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells and can leverage replication-dependent non-polyadenylated histone gene transcription to elucidate cell cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO-seq enables the identification of networks of enhancers and genes. Our results suggest that the bursting of transcription at super-enhancers precedes bursting from associated genes. By imparting insights into the dynamic nature of global transcription and the origin and propagation of transcription signals, we demonstrate the ability of scGRO-seq to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression.

Asunto(s)

Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , ARN , Análisis de Secuencia de ARN , Análisis de Expresión Génica de una Sola Célula , Transcripción Genética , Animales , Humanos , Ratones , Ciclo Celular/genética , Química Clic/métodos , ARN Polimerasas Dirigidas por ADN/análisis , ARN Polimerasas Dirigidas por ADN/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Histonas/metabolismo , Regiones Promotoras Genéticas/genética , ARN/análisis , ARN/biosíntesis , ARN/genética , Análisis de Secuencia de ARN/métodos , Análisis de Expresión Génica de una Sola Célula/métodos , Factores de Tiempo

RESUMEN

The agricultural fungicide cymoxanil (CMX) is commonly used in the treatment of plant pathogens, such as Phytophthora infestans. Although the use of CMX is widespread throughout the agricultural industry and internationally, the exact mechanism of action behind this fungicide remains unclear. Therefore, we sought to elucidate the biocidal mechanism underlying CMX. This was accomplished by first performing a large-scale chemical-genomic screen comprising the 4000 haploid non-essential gene deletion array of the yeast Saccharomyces cerevisiae. We found that gene families related to de novo purine biosynthesis and ribonucleoside synthesis were enriched in the presence of CMX. These results were confirmed through additional spot-test and colony counting assays. We next examined whether CMX affects RNA biosynthesis. Using qRT-PCR and expression assays, we found that CMX appears to target RNA biosynthesis possibly through the yeast dihydrofolate reductase (DHFR) enzyme Dfr1. To determine whether DHFR is a target of CMX, we performed an in-silico molecular docking assay between CMX and yeast, human, and P. infestans DHFR. The results suggest that CMX directly interacts with the active site of all tested forms of DHFR using conserved residues. Using an in vitro DHFR activity assay we observed that CMX inhibits DHFR activity in a dose-dependent relationship.

Asunto(s)

Fungicidas Industriales , Simulación del Acoplamiento Molecular , Proteínas de Saccharomyces cerevisiae , Tetrahidrofolato Deshidrogenasa , Humanos , Antagonistas del Ácido Fólico/farmacología , Fungicidas Industriales/farmacología , ARN/biosíntesis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/genética

RESUMEN

One of the significant challenges during the purification and characterization of antimicrobial peptides (AMPs) from Bacillus sp. is the interference of unutilized peptides from complex medium components during analytical procedures. In this study, a semi-synthetic medium was devised to overcome this challenge. Using a genetic algorithm, the production medium of AMP is optimized. The parent organism, Bacillus licheniformis MCC2514, produces AMP in very small quantities. This AMP is known to inhibit RNA biosynthesis. The findings revealed that lactose, NH4Cl and NaNO3 were crucial medium constituents for enhanced AMP synthesis. The potency of the AMP produced was studied using bacterium, Kocuria rhizophila ATCC 9341. The AMP produced from the optimized medium was eightfold higher than that produced from the unoptimized medium. Furthermore, activity was increased by 1.5-fold when cultivation conditions were standardized using the optimized medium. Later, AMP was produced in a 5 L bioreactor under controlled conditions, which led to similar results as those of shake-flask production. The mode of action of optimally produced AMP was confirmed to be inhibition of RNA biosynthesis. Here, we demonstrate that improved production of AMP is possible with the developed semi-synthetic medium recipe and could help further AMP production in an industrial setup.

Asunto(s)

Algoritmos , Bacillus licheniformis , Medios de Cultivo , Bacillus licheniformis/metabolismo , Bacillus licheniformis/genética , Péptidos Antimicrobianos/biosíntesis , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , ARN/biosíntesis , Reactores Biológicos

RESUMEN

Transcription initiation is a complex process, and its mechanism is incompletely understood. We determined the structures of de novo transcribing complexes TC2 to TC17 with RNA polymerase II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides in length, respectively. Connecting these structures generated a movie and a working model. As initially synthesized RNA grows, general transcription factors (GTFs) remain bound to the promoter and the transcription bubble expands. Nucleoside triphosphate (NTP)-driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may promote the transition from initially transcribing complexes (ITCs) (TC2 to TC9) to early elongation complexes (EECs) (TC10 to TC17). Our study shows dynamic processes of transcription initiation and reveals why ITCs require GTFs and bubble expansion for initial RNA synthesis, whereas EECs need GTF dissociation from the promoter and bubble collapse for promoter escape.

Asunto(s)

ARN , Factores Generales de Transcripción , Iniciación de la Transcripción Genética , ARN Polimerasas Dirigidas por ADN/química , ARN/biosíntesis , ARN Polimerasa II/química , Factores Generales de Transcripción/metabolismo , Humanos , Animales , Sus scrofa , Microscopía por Crioelectrón , Películas Cinematográficas

RESUMEN

Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the transition to the elongation phase. Mitochondria recruit a single-subunit RNAP and one or two auxiliary factors to initiate transcription. Previous studies have revealed the molecular architectures of yeast1 and human2 mitochondrial RNAP initiation complexes (ICs). Here we provide a comprehensive, stepwise mechanism of transcription initiation by solving high-resolution cryogenic electron microscopy (cryo-EM) structures of yeast mitochondrial RNAP and the transcription factor Mtf1 catalysing two- to eight-nucleotide RNA synthesis at single-nucleotide addition steps. The growing RNA-DNA is accommodated in the polymerase cleft by template scrunching and non-template reorganization, creating stressed intermediates. During early initiation, non-template strand scrunching and unscrunching destabilize the short two- and three-nucleotide RNAs, triggering abortive synthesis. Subsequently, the non-template reorganizes into a base-stacked staircase-like structure supporting processive five- to eight-nucleotide RNA synthesis. The expanded non-template staircase and highly scrunched template in IC8 destabilize the promoter interactions with Mtf1 to facilitate initiation bubble collapse and promoter escape for the transition from initiation to the elongation complex (EC). The series of transcription initiation steps, each guided by the interplay of multiple structural components, reveal a finely tuned mechanism for potential regulatory control.

Asunto(s)

Mitocondrias , Saccharomyces cerevisiae , Iniciación de la Transcripción Genética , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/ultraestructura , Mitocondrias/enzimología , Mitocondrias/genética , Mitocondrias/ultraestructura , Nucleótidos/metabolismo , ARN/biosíntesis , ARN/ultraestructura , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Microscopía por Crioelectrón , ADN/metabolismo , ADN/ultraestructura

RESUMEN

T7 RNA polymerase is commonly used to synthesize large quantities of RNA for a wide variety of applications, from basic science to mRNA therapeutics. This in vitro system, while showing high fidelity in many ways, is also well known for producing longer than encoded RNA products, particularly under high-yield reaction conditions. Specifically, the resulting product pool is contaminated by an often disperse collection of longer cis-primed extension products. In addition to reducing yield via the conversion of correctly encoded RNA to longer products, self-primed extension generates partially double-stranded RNAs that can trigger the innate immune response. Extensive and low-yield purifications are then required to produce therapeutic RNA. Under high-yield conditions, accumulating concentrations of RNA effectively compete with promoter DNA for polymerase binding, driving self-primed extension at the expense of correct initiation. In the current work, we introduce a simple and novel modification in the DNA to strengthen promoter binding, shifting the balance back toward promoter-driven synthesis and so dramatically reducing self-primed extension. The result is higher yield of the encoded RNA at the outset and reduced need for extensive purifications. The approach can readily be applied to the synthesis of mRNA-length products under high-yield conditions.

Asunto(s)

ARN , Transcripción Genética , ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Regiones Promotoras Genéticas , ARN Bicatenario , ARN Mensajero/genética , ARN/biosíntesis

RESUMEN

Bacterial small RNAs (sRNAs) that regulate gene expression have been engineered for uses in synthetic biology and metabolic engineering. Here, we designed a novel non-Hfq-dependent sRNA scaffold that uses a modifiable 20 nucleotide antisense binding region to target mRNAs selectively and influence protein expression. The system was developed for regulation of a fluorescent reporter in vivo using Escherichia coli, but the system was found to be more responsive and produced statistically significant results when applied to protein synthesis using in vitro cell-free systems (CFS). Antisense binding sequences were designed to target not only translation initiation regions but various secondary structures in the reporter mRNA. Targeting a high-energy stem loop structure and the 3' end of mRNA yielded protein expression knock-downs that approached 70%. Notably, targeting a low-energy stem structure near a potential RNase E binding site led to a statistically significant 65% increase in protein expression (p < 0.05). These results were not obtainable in vivo, and the underlying mechanism was translated from the reporter system to achieve better than 75% increase in recombinant diaphorase expression in a CFS. It is possible the designs developed here can be applied to improve/regulate expression of other proteins in a CFS.

Asunto(s)

Sistema Libre de Células , ARN , Biología Sintética , Dihidrolipoamida Deshidrogenasa/metabolismo , Regulación de la Expresión Génica , Técnicas In Vitro , ARN/biosíntesis , ARN/metabolismo , Estabilidad del ARN , Biología Sintética/métodos , Análisis de Varianza

RESUMEN

Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation1,2. Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1. The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function.

Asunto(s)

Proteínas de Ciclo Celular , Daño del ADN , Reparación del ADN , Estrés Oxidativo , Regiones Promotoras Genéticas , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Genes , Prueba de Complementación Genética , Mitosis , Mutación , Estrés Oxidativo/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Poli ADP Ribosilación , Regiones Promotoras Genéticas/genética , ARN/biosíntesis , ARN/genética , ARN Polimerasa II/metabolismo , Huso Acromático/metabolismo , Sitio de Iniciación de la Transcripción

RESUMEN

The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways1-4 such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα-primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα-primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα-primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα-primase into two separate domains and effectively arranges them in RNA-DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA-DNA primers by Polα-primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα-primase PIC and activation are also revealed in this study.

Asunto(s)

ADN Primasa , Complejo Shelterina , Telómero , Moldes Genéticos , ADN/metabolismo , ADN Primasa/química , ADN Primasa/metabolismo , Cartilla de ADN/biosíntesis , Replicación del ADN , Humanos , Dominios Proteicos , ARN/biosíntesis , ARN/metabolismo , Complejo Shelterina/química , Complejo Shelterina/metabolismo , Especificidad por Sustrato , Telómero/química , Telómero/genética , Telómero/metabolismo

RESUMEN

Gene regulation in the human genome is controlled by distal enhancers that activate specific nearby promoters1. A proposed model for this specificity is that promoters have sequence-encoded preferences for certain enhancers, for example, mediated by interacting sets of transcription factors or cofactors2. This 'biochemical compatibility' model has been supported by observations at individual human promoters and by genome-wide measurements in Drosophila3-9. However, the degree to which human enhancers and promoters are intrinsically compatible has not yet been systematically measured, and how their activities combine to control RNA expression remains unclear. Here we design a high-throughput reporter assay called enhancer × promoter self-transcribing active regulatory region sequencing (ExP STARR-seq) and applied it to examine the combinatorial compatibilities of 1,000 enhancer and 1,000 promoter sequences in human K562 cells. We identify simple rules for enhancer-promoter compatibility, whereby most enhancers activate all promoters by similar amounts, and intrinsic enhancer and promoter activities multiplicatively combine to determine RNA output (R2 = 0.82). In addition, two classes of enhancers and promoters show subtle preferential effects. Promoters of housekeeping genes contain built-in activating motifs for factors such as GABPA and YY1, which decrease the responsiveness of promoters to distal enhancers. Promoters of variably expressed genes lack these motifs and show stronger responsiveness to enhancers. Together, this systematic assessment of enhancer-promoter compatibility suggests a multiplicative model tuned by enhancer and promoter class to control gene transcription in the human genome.

Asunto(s)

Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Elementos de Facilitación Genéticos/genética , Humanos , Regiones Promotoras Genéticas/genética , ARN/biosíntesis , ARN/genética , Factores de Transcripción/metabolismo

RESUMEN

In biological systems, the synthesis of nucleic acids, such as DNA and RNA, is catalyzed by enzymes in various aqueous solutions. However, substrate specificity is derived from the chemical properties of the residues, which implies that perturbations of the solution environment may cause changes in the fidelity of the reaction. Here, we investigated non-promoter-based synthesis of RNA using T7 RNA polymerase (T7 RNAP) directed by an RNA template in the presence of polyethylene glycol (PEG) of various molecular weights, which can affect polymerization fidelity by altering the solution properties. We found that the mismatch extensions of RNA propagated downstream polymerization. Furthermore, PEG promoted the polymerization of non-complementary ribonucleoside triphosphates, mainly due to the decrease in the dielectric constant of the solution. These results indicate that the mismatch extension of RNA-dependent RNA polymerization by T7 RNAP is driven by the stacking interaction of bases of the primer end and the incorporated nucleotide triphosphates (NTP) rather than base pairing between them. Thus, proteinaceous RNA polymerase may display different substrate specificity with changes in dielectricity caused by molecular crowding conditions, which can result in increased genetic diversity without proteinaceous modification.

Asunto(s)

ARN Polimerasas Dirigidas por ADN/química , ARN/biosíntesis , Proteínas Virales/química , Emparejamiento Base , ARN Polimerasas Dirigidas por ADN/metabolismo , Variación Genética , Polimerizacion , ARN/genética , Ribonucleósidos/química , Ribonucleósidos/metabolismo , Soluciones , Especificidad por Sustrato , Proteínas Virales/metabolismo

RESUMEN

Inflammatory memory involves the molecular and cellular 'reprogramming' of innate immune cells following exogenous stimuli, leading to non-specific protection against subsequent pathogen exposure. This phenomenon has now also been described in non-hematopoietic cells, such as human fetal and adult endothelial cells. In this study we mapped the cell-specific DNA methylation profile and the transcriptomic remodelling during the establishment of inflammatory memory in two distinct fetal endothelial cell types - a progenitor cell (ECFC) and a differentiated cell (HUVEC) population. We show that both cell types have a core transcriptional response to an initial exposure to a viral-like ligand, Poly(I:C), characterised by interferon responsive genes. There was also an ECFC specific response, marked by the transcription factor ELF1, suggesting a non-canonical viral response pathway in progenitor endothelial cells. Next, we show that both ECFCs and HUVECs establish memory in response to an initial viral exposure, resulting in an altered subsequent response to lipopolysaccharide. While the capacity to train or tolerize the induction of specific sets of genes was similar between the two cell types, the progenitor ECFCs show a higher capacity to establish memory. Among tolerized cellular pathways are those involved in endothelial barrier establishment and leukocyte migration, both important for regulating systemic immune-endothelial cell interactions. These findings suggest that the capacity for inflammatory memory may be a common trait across different endothelial cell types but also indicate that the specific downstream targets may vary by developmental stage.

Asunto(s)

Metilación de ADN , Células Progenitoras Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Inflamación/patología , Transcriptoma , Animales , Separación Celular , Células Cultivadas , Células Progenitoras Endoteliales/efectos de los fármacos , Feto/citología , Regulación de la Expresión Génica/efectos de los fármacos , Ontología de Genes , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Recién Nacido , Inflamación/embriología , Inflamación/genética , Inflamación/inmunología , Lipopolisacáridos/farmacología , Ratones , Subfamília D de Receptores Similares a Lectina de las Células NK/biosíntesis , Subfamília D de Receptores Similares a Lectina de las Células NK/genética , Proteínas Nucleares/metabolismo , Poli I-C/farmacología , ARN/biosíntesis , ARN/genética , Factores de Transcripción/metabolismo

RESUMEN

Vibrio cholerae represents a constant threat to public health, causing widespread infections, especially in developing countries with a significant number of fatalities and serious complications every year. The standard treatment by oral rehydration does not eliminate the source of infection, while increasing antibiotic resistance among pathogenic V. cholerae strains makes the therapy difficult. Thus, we assessed the antibacterial potential of plant-derived phytoncides, isothiocyanates (ITC), against V. cholerae O365 strain. Sulforaphane (SFN) and 2-phenethyl isothiocyanate (PEITC) ability to inhibit bacterial growth was assessed. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values indicate that these compounds possess antibacterial activity and are also effective against cells growing in a biofilm. Tested ITC caused accumulation of stringent response alarmone, ppGpp, which indicates induction of the global stress response. It was accompanied by bacterial cytoplasm shrinkage, the inhibition of the DNA, and RNA synthesis as well as downregulation of the expression of virulence factors. Most importantly, ITC reduced the toxicity of V. cholerae in the in vitro assays (against Vero and HeLa cells) and in vivo, using Galleria mellonella larvae as an infection model. In conclusion, our data indicate that ITCs might be considered promising antibacterial agents in V. cholerae infections.

Asunto(s)

Antibacterianos/farmacología , Cólera/dietoterapia , Isotiocianatos/farmacología , Mariposas Nocturnas/microbiología , Sulfóxidos/farmacología , Vibrio cholerae/efectos de los fármacos , Animales , Biopelículas/efectos de los fármacos , Línea Celular , Chlorocebus aethiops , ADN/biosíntesis , Modelos Animales de Enfermedad , Guanosina Tetrafosfato/biosíntesis , Células HeLa , Humanos , Pruebas de Sensibilidad Microbiana , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , ARN/biosíntesis , Células Vero , Vibrio cholerae/patogenicidad , Virulencia/efectos de los fármacos , Factores de Virulencia/biosíntesis

RESUMEN

CRISPR-Cas9 sgRNA libraries have transformed functional genetic screening and have enabled several innovative methods that rely on simultaneously targeting numerous genetic loci. Such libraries could be used in a vast number of biological systems and in the development of new technologies, but library generation is hindered by the cost, time, and sequence data required for sgRNA library synthesis. Here, we describe a rapid enzymatic method for generating robust, variant-matched libraries from any source of cDNA in under 3 h. This method, which we have named SLALOM, utilizes a custom sgRNA scaffold sequence and a novel method for detaching oligonucleotides from solid supports by a strand displacing polymerase. With this method, we constructed libraries targeting the E. coli genome and the transcriptome of developing zebrafish hearts, demonstrating its ability to expand the reach of CRISPR technology and facilitate methods requiring custom libraries.

Asunto(s)

Sistemas CRISPR-Cas , Animales , Proteínas Asociadas a CRISPR , Enzimas de Restricción del ADN , ADN Polimerasa Dirigida por ADN , Escherichia coli/genética , Colorantes Fluorescentes , Técnicas Genéticas , Genoma , Proteínas Fluorescentes Verdes , Humanos , Miocardio/metabolismo , Oligonucleótidos , ARN/biosíntesis , Transcriptoma , Pez Cebra

RESUMEN

Herein, Broccoli/mCherry and an EGFP/mCherry dual-color fluorescent reporting systems have been established to quantify the promoter activity at transcription and translation levels in eukaryotic cells. Based on those systems, four commonly used promoters (CMV and SV40 of Pol II and U6, H1 of Pol III) were accurately evaluated at both the transcriptional and translational levels by combining accurate protein and RNA quantification. Furthermore, we verified that Pol III promoters can induce proteins expression, and Pol II promoter can be applied to express RNA molecules with defined length by combining a self-cleaving ribozyme and an artificial poly(A) tail. The dual-color fluorescence reporting systems described here could play a significant role in evaluating other gene expression regulators for gene therapy.

Asunto(s)

Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Luminiscentes/genética , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , ARN/biosíntesis , Transcripción Genética , Animales , Citomegalovirus/genética , Citomegalovirus/metabolismo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Proteínas Luminiscentes/metabolismo , Ratones , Células 3T3 NIH , ARN/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Virus 40 de los Simios/genética , Virus 40 de los Simios/metabolismo , Proteína Fluorescente Roja

RESUMEN

The catalytic trigger loop (TL) in RNA polymerase (RNAP) alternates between unstructured and helical hairpin conformations to admit and then contact the NTP substrate during transcription. In many bacterial lineages, the TL is interrupted by insertions of two to five surface-exposed, sandwich-barrel hybrid motifs (SBHMs) of poorly understood function. The 188-amino acid, two-SBHM insertion in Escherichia coli RNAP, called SI3, occupies different locations in elongating, NTP-bound, and paused transcription complexes, but its dynamics during active transcription and pausing are undefined. Here, we report the design, optimization, and use of a Cys-triplet reporter to measure the positional bias of SI3 in different transcription complexes and to determine the effect of restricting SI3 movement on nucleotide addition and pausing. We describe the use of H2O2 as a superior oxidant for RNAP disulfide reporters. NTP binding biases SI3 toward the closed conformation, whereas transcriptional pausing biases SI3 toward a swiveled position that inhibits TL folding. We find that SI3 must change location in every round of nucleotide addition and that restricting its movements inhibits both transcript elongation and pausing. These dynamics are modulated by a crucial Phe pocket formed by the junction of the two SBHM domains. This SI3 Phe pocket captures a Phe residue in the RNAP jaw when the TL unfolds, explaining the similar phenotypes of alterations in the jaw and SI3. Our findings establish that SI3 functions by modulating TL folding to aid transcriptional regulation and to reset secondary channel trafficking in every round of nucleotide addition.

Asunto(s)

ARN Polimerasas Dirigidas por ADN/metabolismo , ARN/biosíntesis , Transcripción Genética/fisiología , Dominio Catalítico , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , ARN Bacteriano/genética , Transcripción Genética/genética

RESUMEN

Inducing cardiac myocytes to proliferate is considered a potential therapy to target heart disease, however, modulating cardiac myocyte proliferation has proven to be a technical challenge. The Hippo pathway is a kinase signaling cascade that regulates cell proliferation during the growth of the heart. Inhibition of the Hippo pathway increases the activation of the transcription factors YAP/TAZ, which translocate to the nucleus and upregulate transcription of pro-proliferative genes. The Hippo pathway regulates the proliferation of cancer cells, pluripotent stem cells, and epithelial cells through a cell-cell contact-dependent manner, however, it is unclear if cell density-dependent cell proliferation is a consistent feature in cardiac myocytes. Here, we used cultured human iPSC-derived cardiac myocytes (hiCMs) as a model system to investigate this concept. hiCMs have a comparable transcriptome to the immature cardiac myocytes that proliferate during heart development in vivo. Our data indicate that a dense syncytium of hiCMs can regain cell cycle activity and YAP expression and activity when plated sparsely or when density is reduced through wounding. We found that combining two small molecules, XMU-MP-1 and S1P, increased YAP activity and further enhanced proliferation of low-density hiCMs. Importantly, these compounds had no effect on hiCMs within a dense syncytium. These data add to a growing body of literature that link Hippo pathway regulation with cardiac myocyte proliferation and demonstrate that regulation is restricted to cells with reduced contact inhibition.

Asunto(s)

Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Secuencia de Bases , Recuento de Células , Ciclo Celular/efectos de los fármacos , Diferenciación Celular , División Celular/efectos de los fármacos , Células Cultivadas , Inhibición de Contacto/efectos de los fármacos , Vía de Señalización Hippo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lisofosfolípidos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Polimorfismo de Nucleótido Simple , ARN/biosíntesis , ARN/genética , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacología , Sulfonamidas/farmacología , Factores de Transcripción/fisiología , Proteínas Señalizadoras YAP

RESUMEN

Once it was believed that ribosomal RNA encodes proteins, and GTP hydrolysis supplies the energy for protein synthesis. Everything has changed, when Alexander Spirin joined the science. It turned out that proteins are encoded by a completely different RNA, and GTP hydrolysis only accelerates the process already provided with energy. It was Spirin who first put forward the idea of a Brownian ratchet and explained how and why molecular machines could arise in the RNA world.

Asunto(s)

Guanosina Trifosfato/metabolismo , Biosíntesis de Proteínas , ARN Ribosómico/metabolismo , Bioquímica/historia , Catálisis , ADN Bacteriano/análisis , ARN Polimerasas Dirigidas por ADN/química , Historia del Siglo XX , Hidrólisis , Modelos Moleculares , Pliegue de Proteína , ARN/biosíntesis , Ribosomas/fisiología , U.R.S.S.

Asunto(s)

Vacunas contra la COVID-19 , Industria Farmacéutica/historia , Investigadores/historia , Investigación/historia , Vacunas Sintéticas/historia , Animales , Ensayos Clínicos como Asunto , Industria Farmacéutica/economía , Terapia Genética/historia , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Liposomas , Ratones , Nanopartículas/química , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/terapia , Premio Nobel , Patentes como Asunto/historia , Seudouridina/metabolismo , ARN/biosíntesis , ARN/genética , Estabilidad del ARN , Conejos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Receptores Toll-Like/inmunología , Vacunas de ADN/historia , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/uso terapéutico , Vacunas de ARNm