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| Status |
Public on Apr 23, 2015 |
| Title |
Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
We have generated single-nucleotide resolution, nascent transcription profiles from HeLa cells by developing Native Elongation Transcript sequencing technology for mammalian chromatin (mNET-seq). Our extensive data sets provide a substantial resource to study mammalian nascent transcript profiles. We reveal unanticipated phosphorylation states for RNA polymerase II C-terminal domain (Pol II CTD) at both gene ends. We also observe that following 5’ splice site cleavage by the spliceosome, upstream exon transcripts are tethered to Pol II CTD phosphorylated on the serine 5 position (S5P) which is accumulated over downstream exons. We further show that depletion of termination factors substantially reduces Pol II pausing at gene ends leading to termination defects. Remarkably termination factors play an additional promoter role by restricting non-productive RNA synthesis and redistributing Pol II CTD S2P to promoters. These data demonstrate that CTD phosphorylation is more dynamic and variably distributed across mammalian transcription units than previously envisaged.
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| Overall design |
To monitor nascent RNA within the mammalian Pol II complex, and its association with different CTD phosphorylation states, we employed mNET-seq methodology on HeLa cells, complemented with direct sequencing of chromatin-bound RNA (ChrRNA-seq). mNET-seq was preformed using the antibodies 8WG16, CMA602, CMA603 and CMA601, which are specific for unphosphorylated CTD, Ser2 phosphorylation, Ser5 phosphorylation and all CTD isoforms, respectively. In another experiment, to evaluate the effect of transcription termination factors in nascent RNA production by Pol II, mNET-seq and complemented with ChrRNA-seq was preformed on HeLa cells transfected with siRNA against PTBP1, CPSF73, CstF64+CstF64tau or Xrn2, and the gene profiles were compared with profiles from HeLa transfected with siRNA for Luciferase generated by the same protocol.
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| Contributor(s) |
Nojima T, Gomes T, Grosso AF, Kimura H, Dye MJ, Dhir S, Fonseca MC, Proudfoot NJ |
| Citation(s) |
25910207, 37783853 |
| Submission date |
Aug 12, 2014 |
| Last update date |
Oct 31, 2023 |
| Contact name |
Tomás Gomes |
| E-mail(s) |
tomasgomes@medicina.ulisboa.pt
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| Organization name |
Instituto de Medicina Molecular
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| Street address |
Av. Professor Egas Moniz
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| City |
Lisboa |
| ZIP/Postal code |
1649-028 Lisboa |
| Country |
Portugal |
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| Platforms (2) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
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| Samples (28)
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| Relations |
| BioProject |
PRJNA258089 |
| SRA |
SRP045447 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE60358_RAW.tar |
4.7 Gb |
(http)(custom) |
TAR (of BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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