A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity

Voss, Cristina; Schmitt, Brita; Werner-Simon, Susanne; Lutz, Christian; Werner, S.; Anderl, Jan

doi:10.1186/1471-2199-15-7

Cited by 11 publications

(15 citation statements)

References 28 publications

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“…5c ). Moreover, we used an in vitro RNAP2-dependent run off assay to measure the de novo transcription in vitro 41 . The HeLaScribe DNA template was incubated with nuclear extracts from the isogenic DU145 cells expressing Dox-inducible RBX1 shRNAs, and a transcription reaction without nucleoside triphosphates served as a negative control.…”

Section: Resultsmentioning

confidence: 99%

Heterozygous deletion of chromosome 17p renders prostate cancer vulnerable to inhibition of RNA polymerase II

Liu

et al. 2018

Nat Commun

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Heterozygous deletion of chromosome 17p (17p) is one of the most frequent genomic events in human cancers. Beyond the tumor suppressor TP53, the POLR2A gene encoding the catalytic subunit of RNA polymerase II (RNAP2) is also included in a ~20-megabase deletion region of 17p in 63% of metastatic castration-resistant prostate cancer (CRPC). Using a focused CRISPR-Cas9 screen, we discovered that heterozygous loss of 17p confers a selective dependence of CRPC cells on the ubiquitin E3 ligase Ring-Box 1 (RBX1). RBX1 activates POLR2A by the K63-linked ubiquitination and thus elevates the RNAP2-mediated mRNA synthesis. Combined inhibition of RNAP2 and RBX1 profoundly suppress the growth of CRPC in a synergistic manner, which potentiates the therapeutic effectivity of the RNAP2 inhibitor, α-amanitin-based antibody drug conjugate (ADC). Given the limited therapeutic options for CRPC, our findings identify RBX1 as a potentially therapeutic target for treating human CRPC harboring heterozygous deletion of 17p.

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Section: Resultsmentioning

confidence: 99%

Heterozygous deletion of chromosome 17p renders prostate cancer vulnerable to inhibition of RNA polymerase II

Liu

et al. 2018

Nat Commun

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“…In our study, the uniquely designed primers have been used to solve the problem; our data indicate that the primers successfully detect low levels of RNA transcript by primer extension and qPCR. Our novel method is distinct from those published recently [ 14 , 17 ] and displays high sensitivity, simplicity and low cost. The method will therefore benefit numerous laboratories that need in vitro transcription analysis but cannot easily employ approaches that use radioactive isotopes.…”

Section: Resultsmentioning

confidence: 93%

“…We initially sought to use a similar method to analyze transcription of the E4 reporter vector; however, we were not able to achieve stable results, although the DNA template in our experiment was depleted to a similar level [ 14 ]; it is likely that this is due to the low efficiency of transcription for E4 and ML reporter vectors. Voss et al [ 17 ] have recently reported another approach that can directly detect RNA transcripts in vitro using AffimetricsQuantigene techniques, but it relies on specialist and facilities to complete the experiment. In our study, the uniquely designed primers have been used to solve the problem; our data indicate that the primers successfully detect low levels of RNA transcript by primer extension and qPCR.…”

Section: Resultsmentioning

confidence: 99%

Establishment and Validation of a Non-Radioactive Method for In Vitro Transcription Assay Using Primer Extension and Quantitative Real Time PCR

et al. 2015

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Primer extension-dependent in vitro transcription assay is one of the most important approaches in the research field of gene transcription. However, conventional in vitro transcription assays incorporates radioactive isotopes that cause environmental and health concerns and restricts its scope of application. Here we report a novel non-radioactive method for in vitro transcription analysis by combining primer extension with quantitative real time PCR (qPCR). We show that the DNA template within the transcription system can be effectively eliminated to a very low level by our specially designed approach, and that the primers uniquely designed for primer extension and qPCR can specifically recognize the RNA transcripts. Quantitative PCR data demonstrate that the novel method has successfully been applied to in vitro transcription analyses using the adenovirus E4 and major late promoters. Furthermore, we show that the TFIIB recognition element inhibits transcription of TATA-less promoters using both conventional and nonradioactive in vitro transcription assays. Our method will benefit the laboratories that need to perform in vitro transcription but either lack of or choose to avoid radioactive facilities.

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“…As in other eukaryotes, the RNA Pol II-mediated transcription in C. elegans depends on the binding of transcription factors to specific gene cis-acting sequences [29]. One of the DNA substrates used in our in vitro system is HNDNA, which contains the CMV promoter and other minimal elements required to induce transcription using nuclear extract from mammalian cells [15]. Its core promoter contains all five elements (or similar sequences) typical for C. elegans and was able to induce transcription appropriately with nematode nuclear extract.…”

Section: Discussionmentioning

confidence: 99%

A novel in vitro Caenorhabditis elegans transcription system

Wibisono

Liu

Sun

2020

BMC Mol and Cell Biol

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Background Caenorhabditis elegans is an excellent model organism for biological research, but its contributions to biochemical elucidation of eukaryotic transcription mechanisms have been limited. One of the biggest obstacles for C. elegans biochemical studies is the high difficulty of obtaining functionally active nuclear extract due to its thick surrounding cuticle. A C. elegans in vitro transcription system was once developed by Lichtsteiner and Tjian in the 1990s, but it has not become widely used, most likely because the transcription reactions were re-constituted with nuclear extract from embryos, not from larval or adult worms, and the method of Dounce homogenization used to prepare the nuclear extract could lead to protein instability. Besides Dounce homogenization, several other techniques were developed to break worms, but no transcription reactions were re-constituted following worm disruption using these approaches. A C. elegans transcription system with effective preparation of functionally active nuclear extract from larval or adult worms has yet to be established. Additionally, non-radioactive methods for detecting transcription as alternatives to the conventional radioactive detection also need to be adapted into such an in vitro system. Results By employing Balch homogenization, we achieved effective disruption of larval and adult worms and obtained functionally active nuclear extract through subcellular fractionation. In vitro transcription reactions were successfully re-constituted using such nuclear extract. Furthermore, a PCR-based non-radioactive detection method was adapted into our system to either qualitatively or quantitatively detect transcription. Using this system to assess how pathogen infection affects C. elegans transcription revealed that Pseudomonas aeruginosa infection changes transcription activity in a promoter- or gene-specific manner. Conclusions In this study, we developed an in vitro C. elegans transcription system that re-constitutes transcription reactions with nuclear extract of larval or adult worms and can both qualitatively and quantitatively detect transcription activity using non-radioactive approaches. This in vitro system is useful for biochemically studying C. elegans transcription mechanisms and gene expression regulation. The effective preparation of functionally active nuclear extract in our system fills a technical gap in biochemical studies of C. elegans and will expand the usefulness of this model organism in addressing many biological questions beyond transcription.

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A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity

Cited by 11 publications

References 28 publications

Heterozygous deletion of chromosome 17p renders prostate cancer vulnerable to inhibition of RNA polymerase II

Heterozygous deletion of chromosome 17p renders prostate cancer vulnerable to inhibition of RNA polymerase II

Establishment and Validation of a Non-Radioactive Method for In Vitro Transcription Assay Using Primer Extension and Quantitative Real Time PCR

A novel in vitro Caenorhabditis elegans transcription system

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