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| Status |
Public on Jul 05, 2024 |
| Title |
CRISPRi Screen for Investigating the RORC RNA Switch Mechanisms in Jurkat Cells |
| Organism |
Homo sapiens |
| Experiment type |
Other
|
| Summary |
RNA structural switches are key regulators of gene expression in bacteria, yet their characterization in Metazoa remains limited. Here we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3’UTR of the RORC transcript. In vivo DMS-MaPseq, coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated mRNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally-driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.
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| Overall design |
To investigate how the RORC RNA switch influences gene expression at the molecular level, we performed genome-wide CRISPRi screens in Jurkat T cells using two eGFP reporter constructs: one with the native RORC switch and another with the 77-GA mutation that favors conformation 1.
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| Contributor(s) |
Khoroshkin M, Winters A |
| Citation(s) |
39014073 |
| |
| Submission date |
Apr 28, 2024 |
| Last update date |
Oct 08, 2024 |
| Contact name |
Hani Goodarzi |
| Organization name |
UCSF
|
| Department |
Biochemistry and Biophysics
|
| Street address |
600 16th St, GH S312D
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platforms (1) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
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| Samples (8)
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| This SubSeries is part of SuperSeries: |
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| Relations |
| BioProject |
PRJNA1105615 |
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