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Series GSE110380 Query DataSets for GSE110380
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
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.
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.
Contributor(s) Benhalevy D, Hafner M
Citation(s) 30478324
Submission date Feb 08, 2018
Last update date Feb 25, 2019
Contact name Daniel Benhalevy
Organization name Tel-Aviv University
Department Shmunis School of Biomedicine and Cancer Research
Lab Lab for Cellular RNA Biology
Street address Tel Aviv University, Green Bldg. Room 205
City Tel Aviv
ZIP/Postal code 6997801
Country Israel
Platforms (1)
GPL21290 Illumina HiSeq 3000 (Homo sapiens)
Samples (12)
GSM2989382 Total RNA in negative control cells
GSM2989383 Total RNA in APEX2-NES cells
GSM2989384 Total RNA in H2B-APEX2 cells
BioProject PRJNA433561
SRA SRP132520

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Supplementary file Size Download File type/resource
GSE110380_2016_RNAseqTotalAndBound.xlsx 6.8 Mb (ftp)(http) XLSX
GSE110380_2017_RNAseq_TotalAndBound.xlsx 9.8 Mb (ftp)(http) XLSX
GSE110380_Pooled_LongAndShortFootprintsAnalysis.xlsx 7.2 Mb (ftp)(http) XLSX
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