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| Status |
Public on Nov 26, 2018 |
| Title |
Proximity-CLIP provides a snapshot of occupied cis-acting elements on RNA in different subcellular compartments on a transcriptome-wide scale |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Many cellular RNAs localize to specific subcellular compartments. Currently, methods to systematically study subcellular RNA localization are limited and lagging behind proteomic approaches. Here, we combined APEX2-mediated proximity biotinylation of proteins with PAR-CLIP to simultaneously profile the proteome and the transcriptome bound by RNA binding proteins in any given subcellular compartment. Our approach is fractionation-independent and does not rely on additional RNA manipulation and labeling steps, thus making it easy to apply. Furthermore, it enables to study the locali-zation of RNA processing intermediates, as well as the identification of regulatory RNA cis-acting elements occupied in different cellular compartments. In a proof-of-concept study we studied RNA and protein localization in the nucleus, cytoplasm and at cell-cell interfaces using Proximity-CLIP. These experiments revealed among other in-sights frequent transcriptional readthrough continuing for several kilobases down-stream of the canonical cleavage and polyadenylation site, a differential binding pat-tern of nuclear and cytoplasmic mRNAs, as well as the localization of mRNAs contain-ing 3’UTR CUG sequence elements at cell-cell interfaces, of which many encode regulatory proteins.
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| Overall design |
Proximity-CLIP takes advantage of the occupancy of cellular RNAs by RBPs throughout their life cycle. An APEX2 fusion protein is targeted to a cellular compartment, and cellular RNAs are labeled with 4SU. Cells are incubated with biotin-phenol (BP) for 30 min, before APEX2-mediated BP oxidation is activated by addition of hydrogen peroxide, followed by reaction quenching and 4SU-dependent protein-RNA crosslinking by UV illumination. BP radicals are created locally and either covalently tag proximate proteins or decay. Compartment-specific RNPs are captured by Streptavidin affinity chromatography. Eluted RNA is analyzed either by RNAse treatment followed by small RNA cDNA library preparation of RBP-protected footprints, analogous to PAR-CLIP, or by standard RNAseq. Data also includes RNAseq analysis of the input, i.e. total cell extract RNA prior to Streptavidin chromatography.
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| Contributor(s) |
Benhalevy D, Hafner M |
| Citation(s) |
30478324 |
| Submission date |
Feb 08, 2018 |
| Last update date |
Feb 25, 2019 |
| Contact name |
Daniel Benhalevy |
| E-mail(s) |
benhalevyd@tauex.tau.ac.il
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| Organization name |
Tel-Aviv University
|
| Department |
Shmunis School of Biomedicine and Cancer Research
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| Lab |
Lab for Cellular RNA Biology
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| Street address |
Tel Aviv University, Green Bldg. Room 205
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| City |
Tel Aviv |
| ZIP/Postal code |
6997801 |
| Country |
Israel |
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| Platforms (1) |
| GPL21290 |
Illumina HiSeq 3000 (Homo sapiens) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA433561 |
| SRA |
SRP132520 |
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