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| Status |
Public on Apr 06, 2022 |
| Title |
Screening thousands of transcribed coding and non-coding regions reveals sequence determinants of RNA polymerase II elongation potential. |
| Organism |
Mus musculus |
| Experiment type |
Other
Genome binding/occupancy profiling by high throughput sequencing
|
| Summary |
Organismal growth and development rely on RNA Polymerase II (RNAPII) synthesizing the appropriate repertoire of messenger RNAs (mRNAs) from protein-coding genes. Productive elongation of full-length transcripts is essential for mRNA function, however what determines whether an engaged RNAPII molecule will terminate prematurely or transcribe processively remains poorly understood. Notably, despite a common process for transcription initiation across RNAPII-synthesized RNAs1, RNAPII is highly susceptible to termination when transcribing non-coding RNAs such as upstream antisense RNAs (uaRNAs) and enhancers RNAs (eRNAs)2, suggesting that differences arise during RNAPII elongation. To investigate the impact of transcribed sequence on elongation potential, we developed a method to screen the effects of thousands of INtegrated Sequences on Expression of RNA and Translation using high-throughput sequencing (INSERT-seq). We found that higher AT content in uaRNAs and eRNAs, rather than specific sequence motifs, underlies the propensity for RNAPII termination on these transcripts. Further, we demonstrate that 5’ splice sites exert both splicing-dependent and autonomous, splicing-independent stimulation of processive transcription, even in the absence of polyadenylation signals. Together, our results reveal a potent role for transcribed sequence in dictating gene output at mRNA and non-coding RNA loci, and demonstrate the power of INSERT-seq towards illuminating these contributions.
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| Overall design |
6 groups of INSERT-seq experiments were performed:
- 4 replicates of total-RNA (steady-state) experiment with library at Pou5f1 reporter locus
- 2 replicates of sort-seq experiment with library at Pou5f1 reporter locus
- 2 replicates of run-on RNA (nascent) experiment with library at Pou5f1 reporter locus
- 2 replicates of chromatin-associated RNA experiment with library at Pou5f1 reporter locus
- 3 replicates of total-RNA (steady-state) experiment with library at 4930461G14Rik lincRNA locus
- 3 replicates each of total-RNA (steady-state) experiment with siNT- and siRRP40-treated cells with library at Pou5f1 reporter locus
H3K4me3 ChIP and 20min TT-seq in wild-type F121-9 mESCs were performed in duplicate, four replicates of PRO-seq in wild-type F121-9 mESCs were performed.
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| Contributor(s) |
Vlaming H, Mimoso CA, Martin BJ, Field AR, Adelman K |
| Citation(s) |
35681023 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R01 GM139960 |
Identifying the sequences and factors that govern the fate of elongating RNAPII |
HARVARD UNIVERSITY (MEDICAL SCHOOL) |
Karen L Adelman |
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| |
| Submission date |
Jun 15, 2021 |
| Last update date |
Jul 06, 2022 |
| Contact name |
Hanneke Vlaming |
| E-mail(s) |
hanneke_vlaming@hms.harvard.edu
|
| Organization name |
Harvard Medical School
|
| Department |
Department of Biological Chemistry and Molecular Pharmacology
|
| Lab |
Adelman
|
| Street address |
45 Shattuck St
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
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| Platforms (4)
|
| GPL16417 |
Illumina MiSeq (Mus musculus) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
| GPL21103 |
Illumina HiSeq 4000 (Mus musculus) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (58)
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| Relations |
| BioProject |
PRJNA738070 |
| SRA |
SRP324145 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE178230_200421_mm10_mESC_TTseq_20m_DMSO_BJM_N2_F.bw |
584.7 Mb |
(ftp)(http) |
BW |
| GSE178230_200421_mm10_mESC_TTseq_20m_DMSO_BJM_N2_R.bw |
564.6 Mb |
(ftp)(http) |
BW |
| GSE178230_HV20210611_normalized_14Rikscreens.txt.gz |
498.4 Kb |
(ftp)(http) |
TXT |
| GSE178230_HV20210611_normalized_RRP40KDscreens.txt.gz |
933.6 Kb |
(ftp)(http) |
TXT |
| GSE178230_HV20220110_normalized_WTscreens.txt.gz |
1.8 Mb |
(ftp)(http) |
TXT |
| GSE178230_RAW.tar |
3.4 Mb |
(http)(custom) |
TAR (of TXT) |
| GSE178230_mESC_F121-9.H3K4me3.ChIPseq.50bp.bins.bw |
183.9 Mb |
(ftp)(http) |
BW |
| GSE178230_mm10_mESC_PROseq_mESC_N4_F.bedGraph.gz |
159.7 Mb |
(ftp)(http) |
BEDGRAPH |
| GSE178230_mm10_mESC_PROseq_mESC_N4_R.bedGraph.gz |
176.5 Mb |
(ftp)(http) |
BEDGRAPH |
| GSE178230_sequence_inread_full.fa.gz |
821.1 Kb |
(ftp)(http) |
FA |
| GSE178230_sequences_unspliced_spliced_pooleddata.fa.gz |
878.1 Kb |
(ftp)(http) |
FA |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
| Processed data are available on Series record |
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