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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. Transcriptome-wide profiling of WDR33 binding sites using PAR-CLIP in HEK293 cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Andreas Gruber

PROVIDER: E-GEOD-61123 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

2014-09-25 | GSE61123 | 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

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

Cold induced RNA-binding proteins influence alternative polyadenylation

2013-05-01 | GSE40468 | 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

Cold induced RNA-binding proteins influence alternative polyadenylation

Project description:We employed PAR-CLIP, a recently developed method based on RNA-protein crosslinking, to identify Cirbp and Rbm3 binding sites at transcriptome-level. Genome-wide RNA-seq analysis indicated that cold temperature leads to extensive 3M-bM-^@M-^YUTR lengthening whereas the loss of Cirbp or Rbm3 resulted in 3M-bM-^@M-^YUTR shortening. Combining with PAR-CLIP results, we found that these two RBPs repress the polyadenylation adjacent to their binding sites. Examination of the binding sites of Cirbp and Rbm3 by PAR-CLIP and their influence on the transcriptome by RNA-seq. PARCLIP was performed as in Hafner et al. 2010 Cell 141, 129M-bM-^@M-^S141, with MEFs cell lines stably expressing FLAG-tagged Cirbp and Rbm3. We used 4-thiouridine (4SU) to enhance the crosslink. For RNA-seq, polyadenylated RNA from the mock-transfected cells at 37M-BM-0C or 32M-BM-0C with two replicates, siCirbp-1, siCirbp-2, siRbm3-1 and siRbm3-2 MEFs at 37M-BM-0C were sequenced on Solexa GAII using 76bp single-end kits according to the manufacturerM-bM-^@M-^Ys instructions.

2013-05-01 | E-GEOD-40468 | biostudies-arrayexpress

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

2012-05-15 | GSE37398 | 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

2012-05-15 | GSE37037 | GEO

CPSF controls PAS selection and epidermal differentiation

Project description:Alternative polyadenylation (APA) is a regulatory mechanism that controls gene expression level and function through the usage of different transcription termination sites, but how APA is regulated to influence epidermal tissue differentiation remains under-characterized. Leveraging 3’READS+, we identified prevalent APA in keratinocytes. We further identified a high-confidence list of genes that alters their usages of polyadenylation site (PAS) during keratinocyte differentiation. To begin to understand the mechanisms that modulates PAS selection within a given gene, we identified that the expression of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex is down regulated during keratinocyte differentiation. Suppression of CPSF using RNAi or CRISPRi strongly impaired epidermal regeneration with upregulation of terminal differentiation marker gene expression. Mechanistically, we identified that CPSF suppression decreased the usage of the GRHL3 proximal PAS and upregulated full-length GRHL3 mRNA production. Knockdown of GRHL3 in the context of CPSFi partially restored differentiation marker gene expression. We also found CPSF interacts with RNA-binding proteins to control PAS usage. Thus, our data suggest a model where CPSF binding preference and PAS choices regulate epidermal terminal differentiation.

2020-11-02 | GSE127223 | GEO

The m6A reader CPSF30-L regulates alternative polyadenylation affecting nitrate signaling in Arabidopsis

Project description:As the most ubiquitous internal modification of eukaryotic mRNA, m6A (N6-methyladenosine) modification plays a vital role in almost every aspect of mRNA metabolism. However, the evidence of m6A in regulating the alternative polyadenylation (APA) is limited. APA is controlled by a large protein-RNA complex with many components including CPSF30. Arabidopsis CPSF30 has two isoforms, the longer one named CPSF30-L containing an additional YTH (YT512-B Homology)-domain in the C terminus, which is unique in plant. In this study, we revealed that m6A modification directly regulated APA, by proving the capability of Arabidopsis CPSF30-L YTH domain binding to m6A substrate by using in vitro assay and structural studies. We observed that extensive genes shifted their poly(A) sites in cpsf30-2 and found that numerous genes altered polyadenylation site (PAS) that correlated well with previously identified m6A peaks, indicating that genes carry m6A modification are prone to be regulated by APA. Moreover, we found that several important genes involved in nitrate metabolism are amongst those genes with APA site alteration in cpsf30-2.And we could rescue these APA and nitrate metabolism defects by introducing the wild-type CPSF30-L, but not by m6A-binding defective mutants (W259A, W310A or Y319A), which explained the nitrogen signaling defects of cpsf30-2 discovered previously. Taken together, our results demonstrated that m6A modification could directly regulate APA in Arabidopsis and revealed the function of m6A reader CPSF30-L in nitrate signaling by controlling APA regulation. This study will shed new lights on the roles of m6A modification during RNA 3’-end processing in plant development.

2021-03-16 | GSE145109 | GEO

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