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| Status |
Public on Mar 05, 2020 |
| Title |
iCLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors |
| Organism |
Homo sapiens |
| Experiment type |
Other
|
| Summary |
Alternative splicing generates distinct mRNA isoforms and is crucial for proteome diversity in eukaryotes. The RNA-binding protein (RBP) U2AF65 is central to splicing decisions, as it recognizes 3' splice sites and recruits the spliceosome. We established 'in vitro iCLIP' experiments, in which recombinant RBPs are incubated with long transcripts, to study how U2AF65 recognizes RNA sequences and how this is modulated by trans-acting RBPs. We quantitatively measure U2AF65 affinities at hundreds of binding sites, and compare in vitro and in vivo binding landscapes by mathematical modelling. We find that trans-acting RBPs extensively regulate U2AF65 binding in vivo, including enhanced recruitment to 3' splice sites and clearance of intronic regions. Using machine learning, we identify novel trans-acting RBPs (including FUBP1, BRUNOL6 and PCBP1) that modulate U2AF65 binding and affect splicing outcomes. Our study offers a blueprint for the high-throughput characterization of in vitro mRNP assembly and in vivo splicing regulation.
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| |
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| Overall design |
Comparison of in vitro and in vivo iCLIP data of U2AF65 and other regulatory RBPs
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| |
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| Contributor(s) |
Ebersberger S |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
| Submission date |
Feb 18, 2019 |
| Last update date |
Mar 07, 2020 |
| Contact name |
Stefanie Ebersberger |
| E-mail(s) |
s.ebersberger@imb-mainz.de
|
| Organization name |
Institute of Molecular Biology
|
| Lab |
Koenig Lab
|
| Street address |
Ackermannweg 4
|
| City |
Mainz |
| ZIP/Postal code |
55128 |
| Country |
Germany |
| |
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| Platforms (1) |
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| Samples (8)
|
| GSM3611420 |
imb_koenig_2014_03_15_JKRS23_nxRRM12_WT_1 |
| GSM3611421 |
imb_koenig_2014_03_15_JKRS23_nxRRM12_WT_2 |
| GSM3611422 |
imb_koenig_2014_03_16_JKRS25_U2AF65Nx_1 |
| GSM3611423 |
imb_koenig_2014_03_16_JKRS25_U2AF65Nx_2 |
| GSM3611424 |
imb_koenig_2014_03_15_JKRS23_nxRRM12_MUT_1 |
| GSM3611425 |
imb_koenig_2014_03_15_JKRS23_nxRRM12_MUT_2 |
| GSM3611426 |
imb_koenig_2014_03_16_JKRS25_U2AF65Nx_GS_1 |
| GSM3611427 |
imb_koenig_2014_03_16_JKRS25_U2AF65Nx_GS_2 |
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| This SubSeries is part of SuperSeries: |
| GSE126694 |
An autoinhibitory intramolecular interaction proof-reads RNA recognition by the essential splicing factor U2AF2 |
|
| Relations |
| BioProject |
PRJNA523002 |
| SRA |
SRP186172 |
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