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| Status |
Public on Feb 02, 2024 |
| Title |
Expanded repertoire of RNA-editing-based detection for RNA binding protein interactions (2) |
| Organism |
Homo sapiens |
| Experiment type |
Other
|
| Summary |
RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To expand the collection of rBEs suitable for editing-based RBP-RNA interactome studies, we developed experimental and computational assays to systematically evaluate the editing activities of over thirty A-to-I and C-to-U rBEs in human cells. We identify rBEs that outperform Drosophila ADAR (TRIBE) and mammalian APOBEC1 (STAMP) in the characterization of the binding patterns of sequence-specific and broad-binding RBPs and recommend rBEs that pair effectively in dual-RBP-based applications. We demonstrate that the optimal choice of single or multiple rBEs to fuse to a given or pair of RBPs depends on the editing biases of the rBEs and the binding preferences of the RBPs. Our framework ENGRAVe (Editing nucleotides with general RNA-base-editor variants) reframes the use of and expands the choice of rBEs for the next generation of RBP-RNA target discoveries.
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| Overall design |
The experiment is Poly(A) RNA sequencing of samples corresponding to MS2 coat-protein (MCP)-rBE fusions demonstrating favorable editing profiles on the MCP binding site-containing reporter mRNA. The approach simultaneously detects edits on the reporter mRNA (on-target) and endogenous transcripts (off-target). Editing on endogenous transcripts is considered "off-target" because those RNAs lack MS2 coat protein (MCP) binding sites.
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| Contributor(s) |
Medina-Munoz HC, Kofman E, Jagannatha P, Boyle EA, Yu T, Jones K, Mueller JR, Lykins GD, Ranzau BL, Kohli RM, Komor AC, Yeo GW |
| Citation(s) |
38287010, 39152130 |
| |
| Submission date |
May 15, 2023 |
| Last update date |
Sep 27, 2024 |
| Contact name |
Brian Yee |
| E-mail(s) |
brian.alan.yee@gmail.com
|
| Organization name |
UCSD
|
| Department |
Health
|
| Lab |
Yeo
|
| Street address |
9500 Gilman Dr
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (30)
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| This SubSeries is part of SuperSeries: |
| GSE232520 |
Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies |
|
| Relations |
| BioProject |
PRJNA972646 |
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