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| Status |
Public on Jun 23, 2021 |
| Title |
ActD timecourse in mESCs |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
mRNAs are generally assumed to be loaded instantly with ribosomes upon entry into the cytoplasm. To measure ribosome density on nascent mRNA, we developed nascent Ribo-Seq (nRibo-Seq) by combining Ribo-Seq with progressive 4-thiouridine labelling. In mouse macrophages, we experimentally determined, for the first time, the lag between the appearance of nascent RNA and its association with ribosomes, which was calculated to be 20 - 22 min for bulk mRNA, and approximated the time it takes for mRNAs to be fully loaded with ribosomes to be 41 - 44 min. Notably, ribosomal loading time is adapted to gene function as rapid loading was observed with highly regulated genes. The lag and ribosomal loading time correlate positively with ORF size and half-life, and negatively with tRNA adaptation indices. Similar results were obtained in mESCs, where the lag between IN and FP was even more pronounced with 35 - 38 min. We validated our measurements after stimulation of macrophages with lipopolysaccharide, where the lag between cytoplasmic and translated mRNA leads to a corresponding uncoupling between input and ribosome-protected fragments. Uncoupling is stronger for mRNAs with long ORFs or half-lives, a finding we also confirmed at the level of protein production by nascent chain proteomics. As a consequence of the lag in ribosome loading, ribosome density measurements are distorted when performed under conditions where mRNA levels are far from steady state expression, and transcriptional changes affect ribosome density in a passive way. This study uncovers an unexpected and considerable lag in ribosome loading, and provides guidelines for the interpretation of Ribo-Seq data taking passive effects on ribosome density into account.
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| Overall design |
Transcriptional shut-off with ActD followed by RNA-Seq to determine mRNA half-lives on a transcriptome-wide scale in mouse embryonic stem cells (mESCs).
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| Contributor(s) |
Schott J, Reitter S, Lindner D, Grosser J, Bruer M, Shenoy A, Geiger T, Mathes A, Dobreva G, Stoecklin G |
| Citation(s) |
34480152 |
| Submission date |
Mar 31, 2021 |
| Last update date |
Sep 08, 2021 |
| Contact name |
Johanna Daniela Schott |
| E-mail(s) |
johanna.schott@medma.uni-heidelberg.de
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| Organization name |
Heidelberg University
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| Department |
Mannheim Institute for Innate Immunoscience
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| Lab |
Stoecklin lab
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| Street address |
Ludolf-Krehl-Str. 13-17
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| City |
Mannheim |
| ZIP/Postal code |
68167 |
| Country |
Germany |
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| Platforms (1) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
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| Samples (4)
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| This SubSeries is part of SuperSeries: |
| GSE155236 |
ActD, Ribo-Seq and nRibo-Seq timecourses in RAW264.7 cells and mESCs |
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| Relations |
| BioProject |
PRJNA718868 |
| SRA |
SRP312928 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE171265_ActD_RNASeq_mESCs.csv.gz |
1.1 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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