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Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter

ABSTRACT: Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter

PROVIDER: PRJNA1018528 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR25931001.fastq.gz	Fastqsanger.gz
	SRR25931002.fastq.gz	Fastqsanger.gz
	SRR25931003.fastq.gz	Fastqsanger.gz
	SRR25931004.fastq.gz	Fastqsanger.gz
	SRR25931005.fastq.gz	Fastqsanger.gz

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Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter (1)

Project description: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.

2024-04-11 | GSE242480 | GEO

Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter (2)

2024-04-11 | GSE243456 | GEO

Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter (1)

Project description:Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter (1)

| PRJNA1013561 | ENA

Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter (2)

Project description:Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter (2)

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Elevated pre-mRNA 3' end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation [QuantSeq]

Project description:Cleavage and polyadenylation (CPA) defines the 3’ end of almost all eukaryotic mRNAs. CPA inhibition, or CPAi, leads to transcriptional readthrough. Here, we show that the CPSF-73 inhibitor JTE-607 globally perturbs gene expression, especially for those with a high GC content and located in high gene density regions. Based on regulated alternative polyadenylation (APA) events, we found that more frequently used CPA sites are inhibited by JTE-607 to a greater extent. Consistently, cells with elevated CPA activities, as indicated by preferential usage of proximal APA sites, display greater transcriptional readthrough and gene expression disturbance upon JTE-607 treatment. Remarkably, overexpression of the core CPA factor FIP1 enhances global CPA activity in the cell and leads to greater JTE-607 sensitivity. Taken together, our data indicate that CPAi selectively impacts genes based on their genomic features and the CPA activity of a cell is a key determinant of sensitivity to CPAi.

2023-06-07 | GSE218555 | GEO

Elevated pre-mRNA 3' end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation [ChrRNA-seq]

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Elevated pre-mRNA 3' end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation [QuantSeq-Pool]

2023-06-07 | GSE231527 | GEO

Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens in a dual fluorescence readthrough reporter

Project description:This SuperSeries is composed of the SubSeries listed below.

2024-04-11 | GSE243457 | GEO

Paf1C plays novel subunit-specific roles in alternative cleavage and polyadenylation

Project description:The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and PolII elongation. Here, we provide the first genome-wide analysis of chromatin occupancy by the entire PAF complex in mammalian cells. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3â ends, a profile that sharply contrasted with the yeast complex. Remarkably, our studies identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription. Moreover, we found that depletion of Paf1C subunits also resulted in the accumulation of RNA polymerase II (PolII) over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits leads to global loss of histone H2B ubiquitylation, but surprisingly, there is little impact of Paf1C depletion on other histone modifications, including the tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and suggest that Paf1C could play a role in protecting transcripts from premature cleavage by preventing PolII accumulation at TSS-proximal pA sites. ChIP-seq, RNA-seq and 3'READS of Paf1C factors in mouse C2C12 myoblast cells

2016-01-15 | E-GEOD-72574 | biostudies-arrayexpress

Uncoupling from transcription protects polyadenylation site cleavage from inhibition by DNA damage

Project description:We studied the regulation of pre-messenger RNA 3'-end processing (i.e., cleavage at polyadenylation sites) in response to ultraviolet C irradiation in human cells.

2023-02-06 | GSE203517 | GEO

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