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| Status |
Public on Mar 10, 2020 |
| Title |
Transcription dynamics regulate poly(A) tails and expression of the RNA degradation machinery to balance mRNA levels |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
To investigate the potential influence of transcription on mRNA stability in mammalian cells, we tested the global relationship between rates of synthesis and decay of mRNAs. We estimated synthesis rates by flash 4sU labeling followed by RNA-seq, and decay rates by treating cells with Actinomycin D (ActD) for 0, 2, 4 and 6 hours and measuring half-lives of all expressed genes by MARS-seq analysis. As short-term perturbations of transcription elongation dynamics diminished mRNA stability, we next wished to examine the effect of extended transcription slowdown. To this end, we treated cells with CPT for 24 hours and profiled mRNA stability in a genome-wide manner using ActD time-course and MARS-seq.
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| Overall design |
For synthesis rates estimation, 2 biological replicates were 4sU labeled in MCF7 cells. RNA was fragmented, biotinylated and 4sU-positive sequences were isolated using streptavidin beads. This RNA was rRNA-depleted and used for Lexogene library preparation without poly(A)-enrichment. For half life estimation, MCF7 cells were subjected to Actinomycin D (ActD) treatment for 0, 2, 4 and 6 hours. After isolation of RNA, external RNA was added as a spike to all samples. To examine effect of long CPT treatment, cells were pre-treated with 6 micromolar CPT for 24 hours, followed by the same ActD time-course.
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| Contributor(s) |
Slobodin B, Bahat A, Sehrawat U, Zuckerman B, Ulitsky I, Dikstein R |
| Citation(s) |
32294471 |
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| Submission date |
Feb 11, 2020 |
| Last update date |
Jun 09, 2020 |
| Contact name |
Binyamin Zuckerman |
| Organization name |
Gladstone Institute
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| Street address |
1650 Owens Street
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| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platforms (1) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (22)
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| Relations |
| BioProject |
PRJNA606034 |
| SRA |
SRP248216 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE145097_half-life_transcription.xlsx |
1.5 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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