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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE242480 | GEO | 2024/04/11

REPOSITORIES: GEO

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

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 | GSE243456 | GEO

Knockdown of CFIm25 or CFIm68 decreases RNA polymerase II pausing and transcription

Project description:Transcription of eukaryotic protein-coding genes by RNA polymerase II (pol II) is highly regulated at initiation, elongation and termination and these steps are coordinated with co-transcriptional processing of the emerging pre-mRNA by capping, splicing, and cleavage and polyadenylation. Polyadenylation (Poly(A)) site recognition, which defines the end of the mRNA, relies on the cleavage and polyadenylation (CPA) complex. It was previously observed that knocking-down proteins of the CPA complex affects not only recognition of the poly(A) site but also results in increased pausing of pol II at the beginning of genes. This finding suggests that the CPA complex plays a role in regulating pol II turnover after transcription initiation. To explore this possibility, we knocked-down two subunits of the cleavage factor I (CFIm), CFIm25 and CFIm68, which are part of the CPA complex and involved in alternative polyadenylation, and performed pol II ChIP-seq. Surprisingly, we found that contrary to the effect of knocking down of other CPA factors, reduction of the level of CFIm25 or CFIm68 decreases pol II pausing and transcription genome-wide in HEK293 cells. These findings indicate that different subunits of the CPA complex have opposite effects on pol II pausing at the start of genes.

2019-06-20 | GSE99955 | GEO

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]

2023-06-07 | GSE218545 | GEO

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

U1 snRNP complex with cleavage and polyadenylation factors controls mRNA transcription termination

Project description:Full-length transcription in the majority of human genes depends on U1 snRNP (U1) to co-transcriptionally suppress transcription-terminating premature 3’-end cleavage and polyadenylation (PCPA) from cryptic polyadenylation signals (PASs) in introns. However, the mechanism of this U1 activity, termed telescripting, is unknown. Here, we captured a complex, comprising U1 and CPA factors (U1–CPAFs), that binds intronic PASs and suppresses PCPA. U1–CPAFs are distinct from U1-spliceosomal complexes; they include CPA’s three main subunits, CFIm, CPSF, and CstF, lack essential splicing factors, and associate with transcription elongation and mRNA export complexes. Telescripting requires U1:pre-mRNA base-pairing, which can be disrupted by U1 antisense oligonucleotide (U1 AMO), triggering PCPA. U1 AMO remodels U1–CPAFs, revealing changes, including recruitment of CPA-stimulating factors, that explain U1–CPAFs’ switch from repressive to activated states. Our findings outline U1 telescripting mechanism and demonstrate U1’s unique role as central-regulator of pre-mRNA processing and transcription.

2019-07-31 | GSE135140 | GEO

Regulation of alternative cleavage and polyadenylation by 3’ end processing and splicing factors

Project description:Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage and polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1, and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly influencing C/P events in 5’ introns and U2 impacting those in efficiently spliced introns. Furthermore, PABPN1 regulates expression of transcripts with pAs near the transcription start site, a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results indicate that the abundance of different C/P factors and splicing factors plays diverse roles in APA, and is relevant to APA regulation in biological conditions. knockdown experiments of 23 C/P factors, 3 splicing factors and U1D in mouse C2C12 myoblast cells

2015-04-01 | E-GEOD-62001 | biostudies-arrayexpress

Integrator enforces the fidelity of transcriptional termination at protein-coding genes

Project description:Integrator regulates the 3’-end processing and termination of multiple classes of noncoding RNAs. Depletion of INTS11, the catalytic subunit of Integrator, or ectopic expression of its catalytic dead enzyme impairs the 3’-end processing and termination of a set of protein coding transcripts termed Integrator-regulated termination (IRT) genes. This defect is manifested by increased RNA polymerase II (RNAPII) readthrough and occupancy of serine-2 phosphorylated RNAPII, de-novo trimethylation of lysine 36 on histone H3, and a compensatory elevation of the cleavage and polyadenylation (CPA) complex in the downstream regions. 3’-RNA-sequnecing reveals that proximal polyadenylation site usage relies on the endonuclease activity of INTS11. The DNA sequence encompassing the transcription end sites of IRT genes feature downstream polyadenylation motifs and an enrichment of GC content that permits the formation of secondary structures within the 3’UTR. Taken together, this study identifies a subset of protein-coding transcripts whose 3’-end processing requires the Integrator complex.

2021-11-10 | GSE169680 | GEO

A compendium of conserved cleavage and polyadenylation events in mammalian genes

Project description:Cleavage and polyadenylation is essential for 3’ end processing of almost all eukaryotic mRNAs. Recent studies have shown widespread alternative cleavage and polyadenylation (APA) events leading to mRNA isoforms with different 3’UTRs and/or coding sequences. Here we present a compendium of conserved cleavage and polyadenylation sites (PASs) in mammalian genes, based on ~1.2 billion 3’ end sequencing reads from over 360 human, mouse and rat samples. We show that ~80% of mammalian mRNA genes contain at least one conserved PAS, and ~50% have conserved APA events. PAS conservation generally reduces promiscuous 3’ end processing, stabling gene expression levels across species. Conservation of APA correlates with gene age, gene expression features, and gene functions. Genes with certain functions, such as cell morphology, cell proliferation, and mRNA metabolism, are particularly enriched with APA events. While tissue-specific genes typically have a low APA rate, brain-specific genes tend to evolve APA. We show enrichment of mRNA destabilizing motifs in alternative 3’UTR sequences, leading to substantial differences in mRNA stability between 3’UTR APA isoforms. Using conserved PASs, we reveal sequence motifs surrounding APA sites and a preference of adenosine at the cleavage site. Mutations of the U-rich motif around the PAS often accompany APA profile changes between species. Analysis of lncRNA PASs indicates a mechanism of PAS fixation involving evolution of A-rich motifs. Taken together, our results present a comprehensive view of PAS evolution in mammals, and a phylogenic understanding of APA functions.

2018-08-08 | GSE111134 | GEO

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