title: 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 cross-linked to such RNAs, as can CPSF30. Transcriptome-wide identification of WDR33 targets by photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) showed that WDR33 binds in and very close to the AAUAAA signal in vivo with high specificity. Thus, our data indicate that the large CPSF subunit participating in recognition of the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies.
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, D-06099 Halle, Germany;
authors.forename: Lars
authors.initials: L
authors.lastname: Schönemann
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, D-06099 Halle, Germany;
authors.forename: Uwe
authors.initials: U
authors.lastname: Kühn
authors.affiliation: Computational and Systems Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
authors.forename: Georges
authors.initials: G
authors.lastname: Martin
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, D-06099 Halle, Germany;
authors.forename: Peter
authors.initials: P
authors.lastname: Schäfer
authors.affiliation: Computational and Systems Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
authors.forename: Andreas R
authors.initials: AR
authors.lastname: Gruber
authors.affiliation: Computational and Systems Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
authors.forename: Walter
authors.initials: W
authors.lastname: Keller
authors.affiliation: Computational and Systems Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
authors.forename: Mihaela
authors.initials: M
authors.lastname: Zavolan
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, D-06099 Halle, Germany; ewahle@biochemtech.uni-halle.de.
authors.forename: Elmar
authors.initials: E
authors.lastname: Wahle
mesh_terms: D039223:Cleavage And Polyadenylation Specificity Factor / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D026723:Polyadenylation; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000378:metabolism; D026721:RNA 3' End Processing / Q000235:genetics*
chemical_list: D039223:Cleavage And Polyadenylation Specificity Factor; D009687:Nuclear Proteins; D021122:Protein Subunits; C486022:WDR33 protein, human
keywords: 3′ end formation; RNA processing; poly(A) polymerase; poly(A) site; polyadenylation