TFIIIB roadblocks control alternative cleavage and polyadenylation site selection
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE311276 | GEO | 2025/12/02
REPOSITORIES: GEO
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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.
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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.
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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.
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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.
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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
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
Project description:About 70% of human genes carry multiple polyadenylation signals, and mRNA 3’end formation is dynamically regulated under different physiological conditions. Global 3’end shortening through alternative polyadenylation (APA) correlates with enhanced cellular proliferation, and 3’ untranslated region (UTR) shortening is a widespread phenomenon in tumour cells, where it appears to enhance tumorigenic properties. However, the mechanisms responsible for this dynamic APA regulation remain incompletely understood. Here we show that transcription factor Sp1 binds directly to RNA in vivo and is a common repressor of distal poly (A) site usage. RNA-sequencing (RNA-seq) analysis identified 2344 genes (36% of total mapped mRNA transcripts) with lengthened 3’UTRs upon Sp1 depletion. Sp1 preferentially binds in vivo within the 3’UTRs of many of these lengthened transcripts and inhibits cleavage at distal sites by interacting physically with subunits of the core cleavage and polyadenylation (CPA) machinery. The 3’UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings reveal an important mechanism for dynamic APA regulation by unraveling a novel function of Sp1.
2022-01-28 | GSE165771 | GEO
Project description:About 70% of human genes carry multiple polyadenylation signals, and mRNA 3’end formation is dynamically regulated under different physiological conditions. Global 3’end shortening through alternative polyadenylation (APA) correlates with enhanced cellular proliferation, and 3’ untranslated region (UTR) shortening is a widespread phenomenon in tumour cells, where it appears to enhance tumorigenic properties. However, the mechanisms responsible for this dynamic APA regulation remain incompletely understood. Here we show that transcription factor Sp1 binds directly to RNA in vivo and is a common repressor of distal poly (A) site usage. RNA-sequencing (RNA-seq) analysis identified 2344 genes (36% of total mapped mRNA transcripts) with lengthened 3’UTRs upon Sp1 depletion. Sp1 preferentially binds in vivo within the 3’UTRs of many of these lengthened transcripts and inhibits cleavage at distal sites by interacting physically with subunits of the core cleavage and polyadenylation (CPA) machinery. The 3’UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings reveal an important mechanism for dynamic APA regulation by unraveling a novel function of Sp1.
2022-01-28 | GSE165739 | GEO
Project description:About 70% of human genes carry multiple polyadenylation signals, and mRNA 3’end formation is dynamically regulated under different physiological conditions. Global 3’end shortening through alternative polyadenylation (APA) correlates with enhanced cellular proliferation, and 3’ untranslated region (UTR) shortening is a widespread phenomenon in tumour cells, where it appears to enhance tumorigenic properties. However, the mechanisms responsible for this dynamic APA regulation remain incompletely understood. Here we show that transcription factor Sp1 binds directly to RNA in vivo and is a common repressor of distal poly (A) site usage. RNA-sequencing (RNA-seq) analysis identified 2344 genes (36% of total mapped mRNA transcripts) with lengthened 3’UTRs upon Sp1 depletion. Sp1 preferentially binds in vivo within the 3’UTRs of many of these lengthened transcripts and inhibits cleavage at distal sites by interacting physically with subunits of the core cleavage and polyadenylation (CPA) machinery. The 3’UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings reveal an important mechanism for dynamic APA regulation by unraveling a novel function of Sp1.
2022-01-28 | GSE165683 | GEO
Project description:Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four, CPSF160, CPSF30, hFip1 and WDR33, are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73 and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV-crosslinked to such RNAs, as can be CPSF30. Transcriptome-wide identification of WDR33 targets by PAR-CLIP shows that WDR33 specifically binds the AAUAAA signal in vivo in contrast to any of the five other CPSF subunits tested before. Thus, our data indicate that the CPSF subunit that is mainly responsible for recognizing the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies. Transcriptome-wide profiling of WDR33 binding sites using PAR-CLIP in HEK293 cells.
2014-09-25 | E-GEOD-61123 | biostudies-arrayexpress