|
| Status |
Public on May 18, 2021 |
| Title |
Functional characterization of splicing regulatory elements |
| Organisms |
Homo sapiens; synthetic construct |
| Experiment type |
Other
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| Summary |
RNA binding protein-RNA interactions mediate a variety of processes including pre-mRNA splicing, translation, decay, polyadenylation and many others. Previous high -throughput studies have characterized general sequence features associated with increased and decreased splicing of certain exons, but these studies are limited by not knowing the mechanisms underlying these associations. Here we utilize ENCODE data from diverse data modalities to identify functional splicing regulatory elements and their associated RNA binding proteins (RBPs). We identify features which make splicing events more sensitive to depletion of RBPs, as well as which RBPs act as splicing regulators sensitive tupon RBP depletion. To analyze the sequence determinants underlying RBP-RNA interactions impacting splicing, we assay tens of thousands of sequence variants in a high-throughput splicing reporter called Vex-seq and confirm a small subset in their endogenous loci using CRISPR base editors. Finally, we leverage other large transcriptomic datasets to confirm the importance of RBPs which we designed experiments around and identify additional RBPs which may act as additional splicing regulators of the exons studied.
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| Overall design |
8 samples are included in this experiments which include Vex-seq libraries and the associated plasmid DNA sequencing libraries. These include two amplicon DNA sequencing of a plasmid libraries, which are the primary and secondary plasmid pools, the latter of which was transfected into 3 biological replicates of K562 or HepG2 cells. These transfected cells were quantified using amplicon RT-PCR sequencing.
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| Contributor(s) |
Adamson SI, Zhan L, Graveley BR |
| Citation missing |
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| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R35 GM118140 |
Complex RNA Processing |
UNIVERSITY OF CONNECTICUT HEALTH CENTER |
Brenton R. Graveley |
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| Submission date |
Jan 13, 2021 |
| Last update date |
May 21, 2021 |
| Contact name |
Scott Adamson |
| E-mail(s) |
adamson@uchc.edu
|
| Organization name |
UConn Health
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| Department |
Genetics and Genome Sciences
|
| Lab |
Graveley
|
| Street address |
400 Farmington Ave
|
| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06032 |
| Country |
USA |
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| Platforms (2) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA691896 |
| SRA |
SRP301592 |
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