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| Status |
Public on Apr 11, 2024 |
| Title |
Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter (1) |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
The majority of messenger RNA precursor (pre-mRNA) undergoes 3' end cleavage and polyadenylation (CPA), termed as to 3’ end processing. This processing is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the CPA factors have been discovered through biochemical and proteomic studies. However, genome-wide genetic identification of CPA factors has been hampered by the lack of a reliable high-throughput method. Here, we developed a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters, GFP and mCherry. This system enables the measurement of 3’ end processing in living cells via flow cytometry analysis. Using this system in combination with a human CRISPR library, we conducted a genome-wide screen for CPA factors. The screens identified most of the well-known CPA factors, including most components of the core CPA machineries (i.e. CPSF, CSTF, CFI, and CFII complexes), the CPA scaffold protein SYMPK, PPP1R10 in PNUTS-PP1 complex, as well as CBLL1 (a subunit of m6A RNA methyltransferase complex). Importantly, the screens also uncovered CCNK/CDK12 as a functional core CPA machinery. Furthermore, the screens identified RPRD1B, an RNA polymerase II (RNAP II) binding protein, as a CPA factor. Further characterization showed that RPRD1B binds RNA and regulates the release of RNAP II at the 3’ end of genes. Thus, this dual fluorescence reporter coupled with CRISPR screen provides a reliable tool for identifying bona fide CAP factors, offering a platform for investigating 3’ end processing in various contexts.
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| Overall design |
Flp-In T-Rex HEK293 cells were infected with the pooled lentiviral genome-wide TKOv3 gRNA library at a MOI of 0.3, and subjected to Fluorescence-activated cell sorting (FACS). The gRNA in mCherry/GFP ratio increased cells was compared to that of unsorted cells each with 4 biological replicates.
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| Contributor(s) |
Ni Z, Ahmed N, Nabeel-Shah S, Guo X, Pu S, Song J, Macron E, Burke GL, Tong AY, Chan K, Ha KH, Blencowe BJ, Moffat J, Greenblatt JF |
| Citation(s) |
38587191 |
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| Submission date |
Sep 06, 2023 |
| Last update date |
Apr 12, 2024 |
| Contact name |
Jack Greenblatt |
| Organization name |
University of Toronto
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| Department |
Donnelly Center
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| Lab |
Greenblatt
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| Street address |
160 College St
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
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| Platforms (1) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
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| Samples (16)
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| This SubSeries is part of SuperSeries: |
| GSE243457 |
Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter |
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| Relations |
| BioProject |
PRJNA1013561 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE242480_CCND2_sortALL_SFA_raw_DESeq2_results_table.tab.gz |
849.4 Kb |
(ftp)(http) |
TAB |
| GSE242480_CPEB2_sortALL_SFA_raw_DESeq2_results_table.tab.gz |
913.1 Kb |
(ftp)(http) |
TAB |
| GSE242480_RAW.tar |
2.3 Mb |
(http)(custom) |
TAR (of TAB) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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