&lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt; Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Baptista, Tiago; Devys, Didier

doi:10.3791/57982-v

Cited by 6 publications

(3 citation statements)

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“…RNA labeling and purification were performed in triplicate as previously described with some minor adjustments 46,93 . Briefly, 20 mL of wildtype and mutant S. cerevisiae cells were grown at 30°C to an OD600 ≈0.8 in YPD.…”

Section: Methodsmentioning

confidence: 99%

The INO80 ATP-dependent chromatin remodelling complex alleviates stalled Polymerase II to promote non-coding RNA transcription termination

Luzzi

Szachnowski

Greener

et al. 2020

Preprint

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RNA quality control and timely termination of aberrant transcription are critical for functional gene expression. Here, we report that in Saccharomyces cerevisiae premature transcription termination of mRNAs is coordinated with the transcriptional elongation process and regulated by the evolutionarily conserved ATP-dependent chromatin remodeling complex INO80. Loss of INO80 sensitizes cells to the transcriptional elongation stress drug 6-azauracil and leads to enhanced pausing of elongating RNA Polymerase II across the genome. Transcriptional pausing positively correlates with premature termination of mRNA transcription and is pronounced proximally to promoters at sites of enhanced histone H3 binding to DNA. Cells with deficient INO80 complex accumulate short, unproductive mRNA transcripts on chromatin and are defective in transcription termination mediated by the Nrd1-Nab3-Sen1 (NNS) complex. We find that loss of INO80 compromises the interaction of the RNA surveillance factor Nab2 with short promoter-proximal mRNA transcripts. INO80 promotes cotranscriptional recruitment of Nab2 to chromatin by enabling its interaction with the histone variant H2A.Z. Finally, inactivation of the histone deacetylase complex Rpd3S/Rco1 reduces promoter-proximal pausing and enhances productive transcription through an NNS-dependent termination site when INO80 is compromised. Our work suggests that, by regulation of H2A.Zcontaining nucleosomes, INO80 orchestrates a mechanism for premature transcription termination, linking RNA quality control to the transcriptional process.

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Section: Methodsmentioning

confidence: 99%

The INO80 ATP-dependent chromatin remodelling complex alleviates stalled Polymerase II to promote non-coding RNA transcription termination

Luzzi

Szachnowski

Greener

et al. 2020

Preprint

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“…4tU labeling and purification of nascent RNA. S. cerevisiae and S. pombe nascent RNA was labeled with 4-thiouracil (4tU) as previously described 75,76 . Briefly, S. cerevisiae cultures were grown in 30 ml of YPD medium at 30 °C to log phase (A 600nm ~ 0.8).…”

Section: Cell Synchronization and Flow Cytometrymentioning

confidence: 99%

Hydroxyurea and inactivation of checkpoint kinase MEC1 inhibit transcription termination and pre-mRNA cleavage at polyadenylation sites in budding yeast

Kaur,

Nagar,

Mehta

et al. 2023

Sci Rep

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The DNA damage response (DDR) is an evolutionarily conserved process essential for cell survival. The transcription changes triggered by DDR depend on the nature of DNA damage, activation of checkpoint kinases, and the stage of cell cycle. The transcription changes can be localized and affect only damaged DNA, but they can be also global and affect genes that are not damaged. While the purpose of localized transcription inhibition is to avoid transcription of damaged genes and make DNA accessible for repair, the purpose and mechanisms of global transcription inhibition of undamaged genes are less well understood. We show here that a brief cell treatment with hydroxyurea (HU) globally inhibits RNA synthesis and transcription by RNA polymerase I, II, and III (RNAPI, RNAPII, and RNAPIII). HU reduces efficiency of transcription termination and inhibits pre-mRNA cleavage at the polyadenylation (pA) sites, destabilizes mRNAs, and shortens poly(A) tails of mRNAs, indicating defects in pre-mRNA 3′ end processing. Inactivation of the checkpoint kinase Mec1p downregulates the efficiency of transcription termination and reduces the efficiency of pre-mRNAs clevage at the pA sites, suggesting the involvement of DNA damage checkpoint in transcription termination and pre-mRNA 3′ end processing.

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“…We and others have used 4sU-seq and 4tUseq to quantify newly synthesized RNA following targeted protein depletion in S. cerevisiae (9,12,(18)(19)(20). These methods involve labeling newly synthesized RNA with 4-thiouridine (4sU) or 4-thiouracil (4tU), followed by biochemical purification of labeled RNA (9,21,22). Thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) is an alternative method that directly quantifies 4sU-or 4tU-labeled RNA in the total RNA pool (7).…”

Section: Introductionmentioning

confidence: 99%

An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion inSaccharomyces cerevisiae

Ridenour,

Donczew

2024

Preprint

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Accurate regulation of gene transcription by RNA polymerase II is essential for the growth and development of eukaryotic cells. Although significant progress has been made in understanding the mechanisms that regulate transcription, many questions remain unanswered. Defining the direct effects of transcriptional regulators is critically important to answering these questions. An effective approach for identifying the direct targets of transcriptional regulators is combining rapid protein depletion and quantification of newly transcribed RNA. The auxin-inducible degron (AID) system and thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) are powerful methods to rapidly degrade a target protein and directly quantify newly transcribed RNA, respectively. Both methods have been widely applied to study transcriptional regulation. To address unresolved questions in transcription, we engineered an end-to-end workflow in Saccharomyces cerevisiae to deplete proteins of interest using the AID system and measure newly transcribed RNA using SLAM-seq. We provide a step-by-step protocol to support rapid implementation and demonstrate that the workflow can help define the direct effects of transcriptional regulators using the BET proteins Bdf1 and Bdf2 as a test case. This workflow will help address outstanding questions underlying the molecular basis of transcription and other biological processes in S. cerevisiae and other systems.

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<em>Saccharomyces cerevisiae</em> Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Cited by 6 publications

References 0 publications

The INO80 ATP-dependent chromatin remodelling complex alleviates stalled Polymerase II to promote non-coding RNA transcription termination

The INO80 ATP-dependent chromatin remodelling complex alleviates stalled Polymerase II to promote non-coding RNA transcription termination

Hydroxyurea and inactivation of checkpoint kinase MEC1 inhibit transcription termination and pre-mRNA cleavage at polyadenylation sites in budding yeast

An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion inSaccharomyces cerevisiae

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