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Reconstitution of CPSF active in polyadenylation: Recognition of the polyadenylation signal by WDR33

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE61123 | GEO | 2014/09/25

SECONDARY ACCESSION(S): PRJNA260289

REPOSITORIES: GEO

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Reconstitution of CPSF active in polyadenylation: Recognition of the polyadenylation signal by WDR33

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

The transcriptional terminator XRN2 and the RNA binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Project description:Alternative cleavage and polyadenylation (APA) of pre-mRNAs yields multiple transcripts differing in their 3' end and its regulation is often aberrant in human cancers, including prostate cancer (PC). In this study, we have uncovered a novel mechanism of APA regulation which impinges on the functional interaction between the 5'-3' exonuclease XRN2 and the A/U-rich motif RNA binding protein Sam68 in PC cells. XRN2 promotes recruitment of Sam68 to its target transcripts, where it competes with the cleavage and polyadenylation specificity factor (CPSF) for the recognition of the AAUAAA core polyadenylation signal (PAS) motif. In particular, the Sam68/XRN2 complex suppresses the usage of strong canonical PASs at distal ends of genes and, consequently, promotes the usage of suboptimal proximal PASs.

2022-08-06 | GSE198872 | GEO

Restrictor synergizes with Symplekin and PNUTS to terminate extragenic transcription

Project description:Transcription termination pathways mitigate the detrimental consequences of unscheduled promiscuous initiation occurring at hundreds of thousands of genomic cis-regulatory elements, thus representing cornerstones of genomic regulation in eukaryotes. Restrictor, a complex conserved from worms to humans and composed of the RNA-binding protein ZC3H4 and of WDR82, a Pol II-carboxyterminal repeat domain (CTD)-interacting protein, is required to suppress long-range, processive transcription at thousands of extragenic sites, with instead comparatively limited effects on gene transcription. Restrictor contains no RNA cleavage subunit and does not coopt known cleavage complexes, indicating alternative termination mechanisms. Here we show that Restrictor-driven termination involves Symplekin, a protein associated with RNA cleavage complexes but also involved in cleavage-independent, phosphatase-dependent termination of non-coding RNAs in yeast. Competition among Restrictor, the cleavage and polyadenylation specificity complex (CPSF) and the histone cleavage complex for a limited pool of Symplekin resulted in mutual regulation, thus revealing unexpected regulatory interactions among multiple transcription termination pathways and a general role of Symplekin in both genic and extragenic cleavage-independent termination pathways.

2023-12-14 | GSE240357 | GEO

Restrictor synergizes with Symplekin and PNUTS to terminate extragenic transcription

2023-12-14 | GSE237459 | GEO

Restrictor synergizes with Symplekin and PNUTS to terminate extragenic transcription

2023-12-14 | GSE237458 | GEO

Restrictor synergizes with Symplekin and PNUTS to terminate extragenic transcription

2023-12-14 | GSE237457 | GEO

Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length: CLIP

Project description:Through alternative polyadenylation, human mRNAs acquire longer or shorter 3' untranslated regions, the latter typically associated with higher transcript stability and increased protein production. To understand the dynamics of polyadenylation site usage, we mapped transcriptome‐wide both binding sites of 3' end processing factors CPSF‐160, CPSF‐100, CPSF‐73, CPSF‐30, Fip1, CstF‐64, CstF-64tau, CF Im25, CF Im59, and CF Im68 and 3' end processing sites in HEK293 cells. We found that although binding sites of these factors generally cluster around the poly(A) sites most frequently used in cleavage, CstF‐64/CstF-64tau and CF Im proteins have much higher positional specificity compared to CPSF components. Knockdown of CF Im68 induced a systematic use of proximal polyadenylation sites, indicating that changes in relative abundance of a single 3' end processing factor can modulate the length of 3' untranslated regions transcriptome-wide, and suggesting a mechanism behind the previously observed increase in tumor cell invasiveness upon CF Im68 knockdown. We performed PAR-CLIP experiments for 3' end processing factors including CPSF-30, CPSF-73, CPSF-100, CPSF-160, Fip1, CF Im25, CF Im59, CF Im68, CstF-64, and CstF-64tau.

2012-05-15 | E-GEOD-37398 | biostudies-arrayexpress

Biosynthesis of histone messenger RNA employs a specific 3' end endonuclease

Project description:During S-phase of the cell cycle production of the core histone proteins is precisely balanced with DNA replication. Metazoan mRNAs encoding replication dependent (RD) histones lack polyA tail normally formed by 3’ end cleavage and coupled polyadenylation of the pre-mRNA. Instead, they undergoes to endonucleolytic cleavage on the 3ʹ side of an RNA hairpin (stem loop) producing mRNA with a 3´-stem loop (SL), which is exported from the nucleus for use in translation. The same endonuclease that is involved in normal protein-coding pre-mRNA cleavage, i.e. cleavage and poyladenylation specificity factor 73 (CPSF73), is proposed to catalyse RD pre-histone mRNA cleavage. Additional factors specific to RD pre-histone mRNA processing, including stem loop binding protein (SLBP) and the U7 small nuclear ribonucleoprotein (U7snRNP) that binds to a histone downstream element (HDE) are thought to be involved in CPSF73 targeting to RD pre-histone mRNA. We report that a different histone specific endonuclease (HSE), which like CPSF73 is a metallo β lactamase (MBL) fold protein, is specific for RD pre-histone mRNA cleavage10,11. Crystallographic and biochemical studies reveal HSE has a di-zinc ion containing active site related to that of CPSF73, but which has distinct overall fold. Notably HSE depletion from cells leads to the production of unprocessed RD pre-histone mRNA due to inefficient 3ʹ end processing. The consequent depletion of core histone proteins correlates with a cell cycle defect due to a delay in entering/progressing through S-phase. HSE thus may represent a new type of S-phase specific cancer target.

2018-12-26 | GSE94686 | GEO

Genome?wide analysis of pre?mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length: A-seq

Project description:Through alternative polyadenylation, human mRNAs acquire longer or shorter 3' untranslated regions, the latter typically associated with higher transcript stability and increased protein production. To understand the dynamics of polyadenylation site usage, we mapped transcriptome?wide both binding sites of 3' end processing factors CPSF?160, CPSF?100, CPSF?73, CPSF?30, Fip1, CstF?64, CstF-64tau, CF Im25, CF Im59, and CF Im68 and 3' end processing sites in HEK293 cells. We found that although binding sites of these factors generally cluster around the poly(A) sites most frequently used in cleavage, CstF?64/CstF-64tau and CF Im proteins have much higher positional specificity compared to CPSF components. Knockdown of CF Im68 induced a systematic use of proximal polyadenylation sites, indicating that changes in relative abundance of a single 3' end processing factor can modulate the length of 3' untranslated regions transcriptome-wide, and suggesting a mechanism behind the previously observed increase in tumor cell invasiveness upon CF Im68 knockdown. 3' ends of transcripts were profiled by high-throughput sequencing in HEK 293 cells under normal conditions, and in HEK 293 cells depleted of 3' end processing factors CF Im 68 and CstF-64.

2012-05-15 | E-GEOD-37037 | biostudies-arrayexpress

Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration and invasion

Project description:Cleavage by the endonuclease CPSF73 and polyadenylation of nascent RNA is an essential step in co-transcriptional mRNA maturation, and abnormal poly(A) site choices have been associated with human diseases, including cancer. Recent work has surprisingly identified CPSF73 as a promising drug target for inhibiting the growth of specific cancers. However, our understanding of the regulation of CPSF73 is still at an early stage. Here, we report that a HECT-like E3 ligase, UBE3D, participates in stabilizing CPFS73 protein by preventing its ubiquitin-mediated degradation by the proteasome. Depletion of UBE3D in both non-cancer cells and cancer cells leads to CPSF73 downregulation, a pre-mRNA cleavage defect and dysregulated gene expression. UBE3D dysfunction or chemical inactivation of CPFS73 inhibited migration and invasion as well as stem cell renewal phenotypes in vitro in triple negative breast cancer cells. Our findings indicate that targeting the pre-mRNA processing nuclease CPSF73 has potential for breast cancer therapy.

2021-11-30 | GSE189746 | GEO

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