Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

Dhoondia, Zuzer; Tarockoff, Ricci; Al-Husini, Nadra; Medler, Scott; Agarwal, Neha; Ansari, Athar

doi:10.3791/55446

Cited by 8 publications

(7 citation statements)

References 12 publications

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“…TRO assay was performed essentially as described in [61]. HEM3, BLM10, and SEN1 genes in wild type, sua7-1 and sua7-1 (A) Overexpression of SSU72 in rpb4∆ cells restores the formation of gene loops to wt levels.…”

Section: Tro Assaysmentioning

confidence: 99%

RNA polymerase II plays an active role in the formation of gene loops through the Rpb4 subunit

Allepuz-Fuster

O’Brien

González-Polo

et al. 2019

Preprint

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words)Gene loops are formed by the interaction of initiation and termination factors occupying the distal ends of a gene during transcription. RNAPII is believed to affect gene looping indirectly owing to its essential role in transcription. The results presented here, however, demonstrate a direct role of RNAPII in gene looping through the Rpb4 subunit. 3C analysis revealed that gene looping is abolished in the rpb4∆ mutant. In contrast to the other looping-defective mutants, rpb4∆ cells do not exhibit a transcription termination defect. RPB4 overexpression, however, rescued the transcription termination and gene looping defect of sua7-1, a mutant of TFIIB. Furthermore, RPB4 overexpression rescued the ssu72-2 gene looping defect, while SSU72 overexpression restored the formation of gene loops in rpb4∆ cells. Interestingly, the interaction of TFIIB with Ssu72 is compromised in rpb4∆ cells. These results suggest that the TFIIB-Ssu72 interaction, which is critical for gene loop formation, is facilitated by Rpb4. We propose that Rpb4 is promoting the transfer of RNAPII from the terminator to the promoter for reinitiation of transcription through TFIIB-Ssu72 mediated gene looping.[14]. Besides activators and TFIIB, TFIIH and Mediator complex also play a critical role in gene loop formation by directly interacting with the CF1 and CPF 3'-end processing/termination factors [12,16]. It is generally believed that RNAPII is not directly involved in formation of gene loops, but is a rather passive player in the process. Since gene looping is a transcription-dependent process, it has been presumed that all mutations that affect transcription will also alter gene looping. Accordingly, gene looping was completely abolished in the rpb1-1 mutant [17].The structure of RNAPII is highly conserved among eukaryotes. In all eukaryotes from yeast to humans, RNAPII is composed of 12 subunits. Only Saccharomyces cerevisiae RNAPII exists in a 10-subunit core and a dissociable subcomplex formed by Rpb4 and Rpb7 [18,19].The 12 subunits of RNAPII have been implicated in different functions during the process of transcription. The two largest subunits, Rpb1 and Rpb2, are required for binding to the DNA template. Rpb1 contains a groove for the entry of deoxyribonucleotides. Rpb7 has two RNA binding domains and plays a pivotal role in mRNA decay [20]. Rpb9 has been implicated in start-site selection, but is dispensable for assembly of the 10-subunit enzyme (Hull et al., 1995). While the two largest subunits, Rpb1 and Rpb2, have well-established roles in the initiation of transcription, a few published reports suggest that some of the RNAPII subunits function in the termination of transcription. The CTD of the largest subunit Rpb1 consists of multiple repeats of the heptapeptide sequence (YSPTSPS). The phosphorylation of Ser2 of the CTD is critical for the recruitment of 3'-end processing factors and for termination of transcription [21,22]. In the crystal structure, the Rpb3/Rpb11 heterodimer lies in close proximity of the RNA exit channel [...

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Section: Tro Assaysmentioning

confidence: 99%

RNA polymerase II plays an active role in the formation of gene loops through the Rpb4 subunit

Allepuz-Fuster

O’Brien

González-Polo

et al. 2019

Preprint

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“…With the development of halogenated nucleoside triphosphates such as 5-bromouridine-5′-triphosphate (BrUTP) which are detected by specific antibodies, a wide range of possible downstream applications emerged ( Gratzner, 1982 ; Dundr and Raska, 1993 ; Jensen et al, 1993 ; Wansink et al, 1993 ; Chang et al, 2000 ). However, BrUTP is not absorbed well by living cells, and thus it has to be applied on isolated nuclei in so-called run-on assays ( Thompson et al, 1997 ; Dhoondia et al, 2017 ), or introduced into cells via transfection, injection, or electroporation ( Waksmundzka and Debey, 2001 ). Molecules such as 5-bromouridine (BrU), 5-iodouridine (IU), or 5-fluorouridine (FU), on the other hand, are efficiently taken up by living organisms.…”

Section: Introductionmentioning

confidence: 99%

Visualization of the Nucleolus Using Ethynyl Uridine

Dvořáčková

Fajkus

2018

Front. Plant Sci.

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Thanks to recent innovative methodologies, key cellular processes such as replication or transcription can be visualized directly in situ in intact tissues. Many studies use so-called click iT chemistry where nascent DNA can be tracked by 5-ethynyl-2′-deoxyuridine (EdU), and nascent RNA by 5-ethynyl uridine (EU). While the labeling of replicating DNA by EdU has already been well established and further exploited in plants, the use of EU to reveal nascent RNA has not been developed to such an extent. In this article, we present a protocol for labeling of nucleolar RNA transcripts using EU and show that EU effectively highlights the nucleolus. The method is advantageous, because the need to prepare transgenic plants expressing fluorescently tagged nucleolar components when the nucleolus has to be visualized can be avoided.

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“…Since a terminator proximal intron has been shown to affect termination in metazoans, and intron-dependent gene looping involves a terminator-intron interaction as well, we next examined if the presence of an intron in general, and that of the terminator-proximal intron in particular affect termination of transcription. We monitored termination by transcription run-on (TRO) assay as described in Dhoondia et al (2017) for IMD4 gene with the intron inserted at three different positions as shown in Figure 6A . TRO assay detects the presence of transcriptionally active polymerases on a gene as shown in Figure 6B .…”

Section: Resultsmentioning

confidence: 99%

“…The strand-specific “Transcription Run-On” (TRO) assay was performed as described in ( Dhoondia et al, 2017 ). The primers used for making cDNA and PCR are described in Supplementary Table S2 .…”

Section: Methodsmentioning

confidence: 99%

Proximity to the Promoter and Terminator Regions Regulates the Transcription Enhancement Potential of an Intron

Dwyer

Agarwal

Gega

et al. 2021

Front. Mol. Biosci.

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An evolutionarily conserved feature of introns is their ability to enhance expression of genes that harbor them. Introns have been shown to regulate gene expression at the transcription and post-transcription level. The general perception is that a promoter-proximal intron is most efficient in enhancing gene expression and the effect diminishes with the increase in distance from the promoter. Here we show that the intron regains its positive influence on gene expression when in proximity to the terminator. We inserted ACT1 intron into different positions within IMD4 and INO1 genes. Transcription Run-On (TRO) analysis revealed that the transcription of both IMD4 and INO1 was maximal in constructs with a promoter-proximal intron and decreased with the increase in distance of the intron from the promoter. However, activation was partially restored when the intron was placed close to the terminator. We previously demonstrated that the promoter-proximal intron stimulates transcription by affecting promoter directionality through gene looping-mediated recruitment of termination factors in the vicinity of the promoter region. Here we show that the terminator-proximal intron also enhances promoter directionality and results in compact gene architecture with the promoter and terminator regions in close physical proximity. Furthermore, we show that both the promoter and terminator-proximal introns facilitate assembly or stabilization of the preinitiation complex (PIC) on the promoter. On the basis of these findings, we propose that proximity to both the promoter and the terminator regions affects the transcription regulatory potential of an intron, and the terminator-proximal intron enhances transcription by affecting both the assembly of preinitiation complex and promoter directionality.

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Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

Cited by 8 publications

References 12 publications

RNA polymerase II plays an active role in the formation of gene loops through the Rpb4 subunit

RNA polymerase II plays an active role in the formation of gene loops through the Rpb4 subunit

Visualization of the Nucleolus Using Ethynyl Uridine

Proximity to the Promoter and Terminator Regions Regulates the Transcription Enhancement Potential of an Intron

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