Nascent RNA analyses: tracking transcription and its regulation

Wissink, Erin M.; Vihervaara, Anniina; Tippens, Nathaniel D.; Lis, John T.

doi:10.1038/s41576-019-0159-6

Cited by 212 publications

(184 citation statements)

References 222 publications

(228 reference statements)

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“…NET-seq captures nascent RNAs bound to transcriptionally engaged RNAPII and sequences the 3'-end of the purified RNAs, thus providing a detailed map of elongating RNAPII across the genome at nucleotide resolution . Although the term "nascent RNAs", has been frequently used in the literature to describe newly synthesized RNAs that are not necessarily associated with RNAPII (Wissink et al, 2019), it is supposed to refer to the RNA associated with the polymerase active centre. We will therefore refer to the transcripts captured by NET-seq as "nascent RNAs".…”

Section: Sensitivity To Transcription Elongation Stress and Accumulatmentioning

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: Sensitivity To Transcription Elongation Stress and Accumulatmentioning

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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“…Regulation of the transcription cycle has been a topic of intense research and has been characterized in detail (Chen, Smith, and Shilatifard 2018;Jonkers and Lis 2015;Shandilya and Roberts 2012;Wissink et al 2019). General transcription factors bind to a nucleosome-free promoter causing conformational changes in the DNA helix that allow the binding of Pol II and concomitant synthesis of nascent RNA from the TSS.…”

Section: Introductionmentioning

confidence: 99%

“…Genome-wide ChIP-seq analysis has shown that Pol II accumulates across the promoter proximal region (~-100-+300) including the TSSs and TPSs of most protein coding genes (Chen, Smith, and Shilatifard 2018;L. Core and Adelman 2019;Fraser, Sehgal, and Darnell 1978;Gariglio, Bellard, and Chambon 1981;Jonkers and Lis 2015;Kim et al 2005;Muse et al 2007;Wissink et al 2019;Zeitlinger et al 2007). It has been proposed that the density of Pol II found at the promoter proximal region represents paused Pol II and the ratio of this to the density of Pol II throughout the remainder of the body of the gene (the pausing index) has been used as a measure of transcriptional pausing (Muse et al 2007;Rahl et al 2010;Zeitlinger et al 2007).…”

Section: Introductionmentioning

confidence: 99%

“…Clearly this interpretation depends on whether the promoter-proximal peak of Pol II observed in ChIP experiments reflects initiation, pausing, or both (Ehrensberger, Kelly, and Svejstrup 2013). Considering that pausing occurs only 30-60nts downstream of initiation, the resolution provided by Pol II ChIP-seq is not best suited to resolve these two events in the transcription cycle (Wissink et al 2019). Furthermore, the level of Pol II bound does not necessarily reflect the level of transcription.…”

Section: Introductionmentioning

confidence: 99%

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Enhancers predominantly regulate gene expression in vivo via transcription initiation

Larke

Nojima

Telenius

et al. 2019

Preprint

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Gene transcription occurs via a cycle of linked events including initiation, promoter proximal pausing and elongation of RNA polymerase II (Pol II). A key question is how do transcriptional enhancers influence these events to control gene expression? Here we have used a new approach to quantify transcriptional initiation and pausing in vivo, while simultaneously identifying transcription start sites (TSSs) and pause-sites (TPSs) from single RNA molecules. When analyzed in parallel with nascent RNA-seq, these data show that differential gene expression is achieved predominantly via changes in transcription initiation rather than Pol II pausing. Using genetically engineered mouse models deleted for specific enhancers we show that these elements control gene expression via Pol II recruitment and/or initiation rather than via promoter proximal pause release. Together, our data show that enhancers, in general, control gene expression predominantly by Pol II recruitment and initiation rather than via pausing.

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“…This approach was included 23 in the INSPEcT Bioconductor package, which can now unveil RNA dynamics from steady state or 24 time course data, with or without the profiling of nascent RNA. 25 Main 26 Since the development of microarrays first, and high-throughput sequencing later on, the 27 investigation of the transcriptional activity of genes has been mostly based on the quantification of 28 total RNA (Mortazavi et al, 2008). While bringing about a revolution in the field of transcriptional 29 regulation, the quantification of absolute and differential expression provides only a glimpse of the 30 complexity of cellular gene expression programs.…”

mentioning

confidence: 99%

Genome-wide dynamics of RNA synthesis, processing and degradation without RNA metabolic labeling

Furlan

Galeota

Gaudio

et al. 2019

Preprint

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11The kinetic rates of RNA synthesis, processing and degradation determine the dynamics of 12 transcriptional regulation by governing both the abundance and the responsiveness to modulations of 13 premature and mature RNA species. The study of RNA dynamics is largely based on the integrative 14 analysis of total and nascent transcription, with the latter being quantified through RNA metabolic 15 labeling. We describe here INSPEcT-, a computational method based on mathematical modeling of 16 intronic and exonic expression, able to derive the dynamics of transcription from steady state or time 17 course profiling of just total RNA, without requiring any information on nascent transcripts. Our 18 approach closely recapitulates the kinetic rates obtained through RNA metabolic labeling, improves 19 the ability to detect changes in transcripts half-lives, reduces the cost and complexity of the 20 experiments, and can be adopted to study experimental conditions where nascent transcription cannot 21 be readily profiled. Finally, we applied INSPEcT-to the characterization of post-transcriptional 22 102 First, these data indicate that measurements of mature RNA are in themselves poorly informative 103 of the real transcriptional state of genes. For example, the detection of a mature RNA is typically 104 taken as indication that the corresponding gene is transcriptionally active. This is not necessarily the 105 4 case for highly stable RNAs, which might persist long after the gene has become silent. Second, these 106 data illustrate how difficult it is to decipher the mechanism responsible for modulating mature RNAs 107 without determining the corresponding RNA dynamics. For example, the modulation of mature RNA 108 species is typically seen as indication of transcriptional regulation, while it could originate from 109 changes in the dynamics of processing and/or degradation, without any change in the rate of synthesis 110 taking place. Ultimately, these data show the necessity to develop methods for the quantification of 111 RNA kinetic rates, in order to fully disclose the mechanisms behind complex responses in gene 112 expression. 113

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Nascent RNA analyses: tracking transcription and its regulation

Cited by 212 publications

References 222 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

Enhancers predominantly regulate gene expression in vivo via transcription initiation

Genome-wide dynamics of RNA synthesis, processing and degradation without RNA metabolic labeling

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