scholarly journals Development and optimization of a novel T7 polymerase-independent Marburg virus minigenome system

2020 ◽  
Author(s):  
Bert Vanmechelen ◽  
Joren Stroobants ◽  
Kurt Vermeire ◽  
Piet Maes
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Types ◽  
Marburg Virus ◽  
Attractive Alternative ◽  
Wide Range ◽  
Compound Screening ◽  
Exogenous Expression ◽  
Screening Assays ◽  
T7 Polymerase

AbstractMarburg virus (MARV) is the only known pathogenic filovirus not belonging to the genus Ebolavirus. Minigenomes have proven a useful tool to study MARV, but all existing MARV minigenomes are dependent on the addition of an exogenous T7 RNA polymerase to drive minigenome expression. However, exogenous expression of a T7 polymerase is not always feasible and acts as a confounding factor in compound screening assays. We have developed an alternative minigenome that is controlled by the natively expressed RNA polymerase II. We demonstrate here the characteristics of this new system and its applicability in a wide range of cell types. Our system shows a clear concentration-dependent activity and outperforms the existing T7 polymerase-based system at higher concentrations, especially in difficult-to-transfect cell lines. In addition, we show that our system can be used for high-throughput compound screening in a 96-well format, thereby providing an attractive alternative to previously developed MARV minigenomes.

scholarly journals Development of a T7-Independent MARV Minigenome System

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 27
Author(s):  
Bert Vanmechelen ◽  
Joren Stroobants ◽  
Kurt Vermeire ◽  
Piet Maes
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Virus Disease ◽  
Ectopic Expression ◽  
Cell Types ◽  
Marburg Virus ◽  
Antiviral Compound ◽  
Wide Range ◽  
Compound Screening ◽  
Minigenome System

Marburg virus (MARV) is the only known pathogenic filovirus that does not belong to the genus Ebolavirus. It causes a severe hemorrhagic fever that is associated with a high mortality rate (>80%). The potential for filoviruses to cause devastating outbreaks, in combination with the lack of licensed therapeutics and vaccines for Marburg virus disease, illustrates the need for more MARV research. However, research involving MARV is hindered by its dependency on access to high-containment laboratories. Virus alternatives such as minigenomes have proven to be a useful tool to study virus replication and transcription at lower biosafety levels, and can be used for antiviral compound screening. All currently available MARV minigenomes are dependent on the addition of an ectopic T7 RNA polymerase that can drive minigenome expression. While this allows for high expression levels, the ectopic expression of a T7 polymerase is not feasible in all cell types, and acts as a confounding factor in compound screening assays. We have developed an alternative MARV minigenome system that is controlled by an RNA polymerase II promoter, which is natively expressed in most mammalian cell types. We show here that this novel minigenome can be used in a wide range of cell types, and can be easily amended to a 96-well format to be used for high-throughput compound screening, thereby providing a valuable alternative to previously developed MARV minigenomes.

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals Pausing sites of RNA polymerase II on actively transcribed genes are enriched in DNA double-stranded breaks

2020 ◽  
Vol 295 (12) ◽  
pp. 3990-4000 ◽  
Author(s):  
Sandeep Singh ◽  
Karol Szlachta ◽  
Arkadi Manukyan ◽  
Heather M. Raimer ◽  
Manikarna Dinda ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Topoisomerase I ◽  
Cell Types ◽  
Dna Breaks ◽  
Transcription Start ◽  
Pol Ii ◽  
Transcription Start Sites

DNA double-stranded breaks (DSBs) are strongly associated with active transcription, and promoter-proximal pausing of RNA polymerase II (Pol II) is a critical step in transcriptional regulation. Mapping the distribution of DSBs along actively expressed genes and identifying the location of DSBs relative to pausing sites can provide mechanistic insights into transcriptional regulation. Using genome-wide DNA break mapping/sequencing techniques at single-nucleotide resolution in human cells, we found that DSBs are preferentially located around transcription start sites of highly transcribed and paused genes and that Pol II promoter-proximal pausing sites are enriched in DSBs. We observed that DSB frequency at pausing sites increases as the strength of pausing increases, regardless of whether the pausing sites are near or far from annotated transcription start sites. Inhibition of topoisomerase I and II by camptothecin and etoposide treatment, respectively, increased DSBs at the pausing sites as the concentrations of drugs increased, demonstrating the involvement of topoisomerases in DSB generation at the pausing sites. DNA breaks generated by topoisomerases are short-lived because of the religation activity of these enzymes, which these drugs inhibit; therefore, the observation of increased DSBs with increasing drug doses at pausing sites indicated active recruitment of topoisomerases to these sites. Furthermore, the enrichment and locations of DSBs at pausing sites were shared among different cell types, suggesting that Pol II promoter-proximal pausing is a common regulatory mechanism. Our findings support a model in which topoisomerases participate in Pol II promoter-proximal pausing and indicated that DSBs at pausing sites contribute to transcriptional activation.

scholarly journals Engineered mini H1 promoters with dedicated RNA Polymerase II or III activity

2020 ◽  
pp. jbc.RA120.015386
Author(s):  
Zongliang Gao ◽  
Yme Ubeles van der Velden ◽  
Minghui Fan ◽  
Cynthia Alyssa van der Linden ◽  
Monique Vink ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Polymerase Activity ◽  
Attractive Alternative ◽  
Pol Ii ◽  
Guide Rnas ◽  
Non Coding Rna ◽  
Pol Iii ◽  
Short Hairpin Rnas

RNA polymerase III promoters such as 7SK, U6 and H1 are widely used for the expression of small non-coding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a non-coding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting the Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly-used H1 promoter in terms of activity and small promoter size, but also concerning safety by exclusive expression of the desired therapeutic transcript (either Pol II or Pol III, but not both).

scholarly journals Role for a Filamentous Nuclear Assembly of IFI16, DNA, and Host Factors in Restriction of Herpesviral Infection

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Philipp E. Merkl ◽  
David M. Knipe
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Nucleus ◽  
Mammalian Cells ◽  
Cell Types ◽  
Restriction Factors ◽  
Structured Illumination Microscopy ◽  
Viral Dna ◽  
Antiviral Responses ◽  
Hsv 1

ABSTRACTSeveral host cell nuclear factors are known to restrict herpes simplex virus 1 (HSV-1) replication, but their mechanisms of action remain to be defined. Interferon-inducible protein 16 (IFI16) and the nuclear domain 10-associated proteins, such as promyelocytic leukemia (PML) protein, localize to input viral genomes, but they are also capable of restricting progeny viral transcription. In this study, we used structured illumination microscopy to show that after HSV DNA replication, IFI16 forms nuclear filamentous structures on DNA within a subset of nuclear replication compartments in HSV-1 ICP0-null mutant virus-infected human cells. The ability to form filaments in different cell types correlates with the efficiency of restriction, and the kinetics of filament formation and epigenetic changes are similar. Thus, both are consistent with the filamentous structures being involved in epigenetic silencing of viral progeny DNA. IFI16 filaments recruit other restriction factors, including PML, Sp100, and ATRX, to aid in the restriction. Although the filaments are only in a subset of the replication compartments, IFI16 reduces the levels of elongation-competent RNA polymerase II (Pol II) in all replication compartments. Therefore, we propose that IFI16 filaments with associated restriction factors that form in replication compartments constitute a “restrictosome” structure that signals incisandtransto silence the progeny viral DNA throughout the infected cell nucleus. The IFI16 filamentous structure may constitute the first known nuclear supramolecular organizing center for signaling in the cell nucleus.IMPORTANCEMammalian cells exhibit numerous strategies to recognize and contain viral infections. The best-characterized antiviral responses are those that are induced within the cytosol by receptors that activate interferon responses or shut down translation. Antiviral responses also occur in the nucleus, yet these intranuclear innate immune responses are poorly defined at the receptor-proximal level. In this study, we explored the ability of cells to restrict infection by assembling viral DNA into transcriptionally silent heterochromatin within the nucleus. We found that the IFI16 restriction factor forms filaments on DNA within infected cells. These filaments recruit antiviral restriction factors to prevent viral replication in various cell types. Mechanistically, IFI16 filaments inhibit the recruitment of RNA polymerase II to viral genes. We propose that IFI16 filaments with associated restriction factors constitute a “restrictosome” structure that can signal to other parts of the nucleus where foreign DNA is located that it should be silenced.

scholarly journals RNA polymerase II pausing as a context-dependent reader of the genome

2016 ◽  
Vol 94 (1) ◽  
pp. 82-92 ◽  
Author(s):  
Adam Scheidegger ◽  
Sergei Nechaev
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Types ◽  
Gene Promoters ◽  
Cell Type ◽  
Pol Ii ◽  
Cell Type Specific ◽  
Pol Ii Pausing ◽  
Context Dependent ◽  
Specific Regulation

The RNA polymerase II (Pol II) transcribes all mRNA genes in eukaryotes and is among the most highly regulated enzymes in the cell. The classic model of mRNA gene regulation involves recruitment of the RNA polymerase to gene promoters in response to environmental signals. Higher eukaryotes have an additional ability to generate multiple cell types. This extra level of regulation enables each cell to interpret the same genome by committing to one of the many possible transcription programs and executing it in a precise and robust manner. Whereas multiple mechanisms are implicated in cell type-specific transcriptional regulation, how one genome can give rise to distinct transcriptional programs and what mechanisms activate and maintain the appropriate program in each cell remains unclear. This review focuses on the process of promoter-proximal Pol II pausing during early transcription elongation as a key step in context-dependent interpretation of the metazoan genome. We highlight aspects of promoter-proximal Pol II pausing, including its interplay with epigenetic mechanisms, that may enable cell type-specific regulation, and emphasize some of the pertinent questions that remain unanswered and open for investigation.

scholarly journals The Transcription of Life: From DNA to RNA

2021 ◽  
Vol 8 ◽  
Author(s):  
Roger D. Kornberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chemical Reactions ◽  
Human Body ◽  
Genetic Information ◽  
Cell Types ◽  
Environmental Information ◽  
Environmental Signals ◽  
Open Questions ◽  
The Future

In the last 50 years, I have dedicated my career to studying fundamental questions in biology. These questions address some of the most basic processes of life, such as “How do cells carrying the same genetic information differentiate into some 200 cell types in the human body?” and “How do cells remodel in response to environmental information?” In this article I will take you on a journey through some of my research into these questions. I will describe my main findings about DNA and its transcription to mRNA through a complex machinery called RNA polymerase II. mRNA is eventually translated into proteins that play a variety of major roles within the organism, including building cells, responding to environmental signals, accelerating chemical reactions, and transmitting signals between distant tissues. Last, I will share with you some fascinating open questions that we are now working on and close with a few tips for you—the scientists of the future.

Autoantibodies as probes for the molecular architecture of the nucleus

1991 ◽  
Vol 49 ◽  
pp. 18-19
Author(s):  
David L. Spector ◽  
Gayle Lark ◽  
Mika Sovak ◽  
Sui Huang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cell ◽  
Cell Types ◽  
Detectable Effect ◽  
Molecular Architecture ◽  
Small Nuclear Ribonucleoprotein ◽  
Highly Sensitive ◽  
Polymerase I ◽  
Nuclear Ribonucleoprotein

Autoantibodies to a variety of nuclear components including DNA, histones, DNA polymerases, DNA topoisomerases, lamins, coiled bodies, RNA polymerase I, and several classes of ribonucleoproteins have been identified in the sera of individuals with autoimmune disorders. We have taken advantage of autoantibodies to several major classes (U1, U2, U4/U6, U5) of small nuclear ribonucleoprotein particles (snRNPs) which play a crucial role in the processing of pre-mRNA molecules. We have used these antibodies to evaluate the organization of factors which participate in pre-mRNA splicing in the mammalian cell nucleus.We have previously shown snRNPs to be localized to discrete immunostained regions, “speckles”, which form a latticework within the interphase nuclei of several mammalian cell types. Since RNA polymerase II, which is involved in the synthesis of pre-mRNA, is highly sensitive to a-amanitin we were interested in the effect of an inhibition of pre-mRNA synthesis on the snRNP distribution pattern. Cells incubated in drug for 5 hours (Fig. 1d) showed no detectable effect on the organization of snRNPs nor any effect on the level of transcription.

scholarly journals Localization of the glucocorticoid receptor in discrete clusters in the cell nucleus

1995 ◽  
Vol 108 (9) ◽  
pp. 3003-3011 ◽  
Author(s):  
B. van Steensel ◽  
M. Brink ◽  
K. van der Meulen ◽  
E.P. van Binnendijk ◽  
D.G. Wansink ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Nucleus ◽  
Glucocorticoid Receptors ◽  
Cell Types ◽  
Activation Of Transcription ◽  
Discrete Domains ◽  
Receptor Clusters ◽  
Antagonist Ru486

The cell nucleus is highly organized. Many nuclear functions are localized in discrete domains, suggesting that compartmentalization is an important aspect of the regulation and coordination of nuclear functions. We investigated the subnuclear distribution of the glucocorticoid receptor, a hormone-dependent transcription factor. By immunofluorescent labeling and confocal microscopy we found that after stimulation with the agonist dexamethasone the glucocorticoid receptor is concentrated in 1,000-2,000 clusters in the nucleoplasm. This distribution was observed in several cell types and with three different antibodies against the glucocorticoid receptor. A similar subnuclear distribution of glucocorticoid receptors was found after treatment of cells with the antagonist RU486, suggesting that the association of the glucocorticoid receptor in clusters does not require transformation of the receptor to a state that is able to activate transcription. By dual labeling we found that most dexamethasone-induced receptor clusters do not colocalize with sites of pre-mRNA synthesis. We also show that RNA polymerase II is localized in a large number of clusters in the nucleus. Glucocorticoid receptor clusters did not significantly colocalize with these RNA polymerase II clusters or with domains containing the splicing factor SC-35. Taken together, these results suggest that most clustered glucocorticoid receptor molecules are not directly involved in activation of transcription.

scholarly journals A unified view of the sequence and functional organization of the human RNA polymerase II promoter

2020 ◽  
Vol 48 (14) ◽  
pp. 7767-7785
Author(s):  
Donal S Luse ◽  
Mrutyunjaya Parida ◽  
Benjamin M Spector ◽  
Kyle A Nilson ◽  
David H Price
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Functional Organization ◽  
Sequence Motif ◽  
Promoter Strength ◽  
Sequence Element ◽  
Pol Ii ◽  
Run Off ◽  
Wide Range

Abstract To better understand human RNA polymerase II (Pol II) promoters in the context of promoter-proximal pausing and local chromatin organization, 5′ and 3′ ends of nascent capped transcripts and the locations of nearby nucleosomes were accurately identified through sequencing at exceptional depth. High-quality visualization tools revealed a preferred sequence that defines over 177 000 core promoters with strengths varying by >10 000-fold. This sequence signature encompasses and better defines the binding site for TFIID and is surprisingly invariant over a wide range of promoter strength. We identified a sequence motif associated with promoter-proximal pausing and demonstrated that cap methylation only begins once transcripts are about 30 nt long. Mapping also revealed a ∼150 bp periodic downstream sequence element (PDE) following the typical pause location, strongly suggestive of a +1 nucleosome positioning element. A nuclear run-off assay utilizing the unique properties of the DNA fragmentation factor (DFF) coupled with sequencing of DFF protected fragments demonstrated that a +1 nucleosome is present downstream of paused Pol II. Our data more clearly define the human Pol II promoter: a TFIID binding site with built-in downstream information directing ubiquitous promoter-proximal pausing and downstream nucleosome location.

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