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| Status |
Public on Sep 13, 2021 |
| Title |
STL-seq reveals distinct pause release and termination kinetics for promoter-proximal paused RNA polymerase II transcripts |
| Organisms |
Drosophila melanogaster; Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
Despite the critical regulatory function of promoter-proximal pausing, the influence of pausing kinetics on transcriptional control remains an active area of investigation. Here, we present Start-TimeLapse-seq (STL-seq), a method that captures the genome-wide kinetics of short, capped RNA turnover and reveals principles of regulation at the pause site. By measuring the rates of release into elongation and premature termination through inhibition of pause release, we determine that pause-release rates are highly variable and most promoter-proximal paused RNA Polymerase II molecules prematurely terminate (~80%). The preferred regulatory mechanism upon a hormonal stimulus (20-hydroxyecdysone) is to influence pause-release rather than termination rates. Transcriptional shutdown occurs concurrently with induction of promoter-proximal termination under hyperosmotic stress but paused transcripts from TATA box-containing promoters remain stable, demonstrating an important role for cis-acting DNA elements in pausing. STL-seq dissects the kinetics of pause release and termination, providing an opportunity to identify mechanisms of transcriptional regulation.
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| Overall design |
RNA isolated from Drosophila S2 cells treated +/- ecdysone hormone, +/- flavopiridol inhibitor, and +/- s4U or with triptolide +s4U was either subjected to oxidative-nucleophilic-aromatic-substitution chemistry and enrichment of short, capped RNA species or biotinylation and enrichment of s4U-labeled RNA followed by oxidative-nucleophilic-aromatic-substitution chemistry. RNA isolated from human HEK293T cells treated +/- KCl and +/- s4U was subjected to oxidative-nucleophilic-aromatic-substitution chemistry and enrichment of short, capped RNA species. Sequencing libraries were prepared from all RNA.
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| Contributor(s) |
Zimmer JT, Rosa-Mercado NA, Canzio D, Steitz JA, Simon MD |
| Citation(s) |
34520723 |
| |
| Submission date |
Feb 04, 2021 |
| Last update date |
Dec 13, 2021 |
| Contact name |
Matthew Simon |
| E-mail(s) |
matthew.simon@yale.edu
|
| Organization name |
Yale University
|
| Department |
MBB
|
| Lab |
Simon Lab
|
| Street address |
West Campus, 100 West Campus Drive, Ste MIC312A
|
| City |
Orange |
| State/province |
CT |
| ZIP/Postal code |
06477 |
| Country |
USA |
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| Platforms (3) |
| GPL21306 |
Illumina HiSeq 4000 (Drosophila melanogaster) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
| GPL25244 |
Illumina NovaSeq 6000 (Drosophila melanogaster) |
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| Samples (32)
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| Relations |
| BioProject |
PRJNA699580 |
| SRA |
SRP304766 |
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