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| Status |
Public on Feb 19, 2021 |
| Title |
Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved AS-NMD pathways |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
Background: Alternative splicing (AS), which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing AS pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses.
Results: RNA immunoprecipitation in tandem (RIPiT) of exon junction complex (EJC) components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compared EJC RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our EJC RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identified thousands of previously unannotated splicing events; while many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis.
Conclusions: Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the NMD-linked AS pathway predominates. EJC RIPiT-Seq also enabled identification of numerous conserved but previously unknown AS-NMD events.
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| Overall design |
We created and sequenced rRNA-depleted whole cell RNA-Seq libraries (84-93 million mate pairs each) wherein the captured fragments were of similar length (220-500 nts) to our previously published EJC RIPiT-Seq libraries (GEO: GSE115788). The new RNA-Seq libraries were generated from cultures (three biological replicates, two technical replicates each) that were (+) or were not (–) subjected to a one hour pre-treatment with harringtonine. Technical replicates were later combined.
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| Contributor(s) |
Kovalak C, Donovan S, Bicknell AA, Metkar M, Moore MJ |
| Citation(s) |
33941243 |
| |
| Submission date |
May 10, 2020 |
| Last update date |
May 12, 2021 |
| Contact name |
Carrie Kovalak |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Massachusetts Medical School
|
| Department |
RNA Therapeutics Institute
|
| Lab |
Melissa Moore
|
| Street address |
365 Plantation Street
|
| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platforms (1) |
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| Samples (12)
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| GSM4543716 |
RNA-Seq (-)Harringtonine bio_rep1 tech_rep1 |
| GSM4543717 |
RNA-Seq (-)Harringtonine bio_rep1 tech_rep2 |
| GSM4543718 |
RNA-Seq (-)Harringtonine bio_rep2 tech_rep1 |
| GSM4543719 |
RNA-Seq (-)Harringtonine bio_rep2 tech_rep2 |
| GSM4543720 |
RNA-Seq (-)Harringtonine bio_rep3 tech_rep1 |
| GSM4543721 |
RNA-Seq (-)Harringtonine bio_rep3 tech_rep2 |
| GSM4543722 |
RNA-Seq (+)Harringtonine bio_rep1 tech_rep1 |
| GSM4543723 |
RNA-Seq (+)Harringtonine bio_rep1 tech_rep2 |
| GSM4543724 |
RNA-Seq (+)Harringtonine bio_rep2 tech_rep1 |
| GSM4543725 |
RNA-Seq (+)Harringtonine bio_rep2 tech_rep2 |
| GSM4543726 |
RNA-Seq (+)Harringtonine bio_rep3 tech_rep1 |
| GSM4543727 |
RNA-Seq (+)Harringtonine bio_rep3 tech_rep2 |
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| Relations |
| BioProject |
PRJNA631548 |
| SRA |
SRP261001 |
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