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Series GSE243456 Query DataSets for GSE243456
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 (2)
Organism Homo sapiens
Experiment type Genome binding/occupancy profiling by high throughput sequencing
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.
Overall design RNA polymerase II (RNAP II) ChIP-seq data of RPRD1B knockout and Scramble control, as well as overexpression of GFP-tagged RPRD1B and GFP control in HEK293 cells (Treated with 1µg/ml doxycycline for 4 days), each with 2 biological replicates and input sample.
Contributor(s) Ni Z, Ahmed N, Nabeel-Shah S, Guo X, Pu S, Song J, Macron E, Burke GL, Yan Tong AH, Chan K, Ha KC, Blencowe BJ, Moffat J, Greenblatt JF
Citation(s) 38587191
Submission date Sep 18, 2023
Last update date Apr 11, 2024
Contact name Jack Greenblatt
Organization name University of Toronto
Department Donnelly Center
Lab Greenblatt
Street address 160 College St
City Toronto
State/province Ontario
ZIP/Postal code M5S 3E1
Country Canada
Platforms (1)
GPL16791 Illumina HiSeq 2500 (Homo sapiens)
Samples (12)
GSM7787420 ChIP-seq of Scramble knockout, replicate 1
GSM7787421 ChIP-seq of Scramble knockout, replicate 2
GSM7787422 ChIP-seq of RPRD1B knockout, replicate 1
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
BioProject PRJNA1018532

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Supplementary file Size Download File type/resource
GSE243456_CRI_RPRD1B_merged_at_TTS_7.pdf 58.0 Kb (ftp)(http) PDF
GSE243456_CriScramble_0_N20_peaks.broadPeak.bed.gz 2.2 Mb (ftp)(http) BED
GSE243456_CriScramble_0_N20_peaks.narrowPeak.bed.gz 2.5 Mb (ftp)(http) BED
GSE243456_GFP_N20_peaks.broadPeak.bed.gz 3.1 Mb (ftp)(http) BED
GSE243456_GFP_N20_peaks.narrowPeak.bed.gz 3.0 Mb (ftp)(http) BED
GSE243456_GFP_RPRD1B_merged_at_TTS_7.pdf 56.0 Kb (ftp)(http) PDF
GSE243456_RPRD1B_Cri_peaks.broadPeak.bed.gz 2.7 Mb (ftp)(http) BED
GSE243456_RPRD1B_Cri_peaks.narrowPeak.bed.gz 3.0 Mb (ftp)(http) BED
GSE243456_RPRD1B_GFP_peaks.broadPeak.bed.gz 2.8 Mb (ftp)(http) BED
GSE243456_RPRD1B_GFP_peaks.narrowPeak.bed.gz 2.9 Mb (ftp)(http) BED
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