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Reconstitution of CPSF Active in Polyadenylation: Recognition of the Polyadenylation Signal by WDR33

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Reconstitution of CPSF Active in Polyadenylation: Recognition of the Polyadenylation Signal by WDR33 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33 Lars Schonemann,€ 1 Uwe Kuhn,€ 1 Georges Martin,2 Peter Schafer,€ 1 Andreas R. Gruber,2 Walter Keller,2 Mihaela Zavolan,2 and Elmar Wahle1 1Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, D-06099 Halle, Germany; 2Computational and Systems Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 39 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. [Keywords: RNA processing; 39 end formation; polyadenylation; poly(A) site; poly(A) polymerase] Supplemental material is available for this article. Received August 13, 2014; revised version accepted September 23, 2014. All eukaryotic mRNAs, with the exception of histone proteins or microRNAs. Thus, there is substantial interest mRNAs, undergo a 39 end maturation step consisting of a in the mechanism of poly(A) site recognition and of specific endonucleolytic cleavage of the precursor fol- alternative poly(A) site choice (Campigli Di Giammartino lowed by polyadenylation of the upstream cleavage et al. 2011; Shi 2012; Elkon et al. 2013; Lianoglou et al. fragment; the downstream fragment is degraded (Wahle 2013; Tian and Manley 2013). and Ruegsegger€ 1999; Zhao et al. 1999; Millevoi and In mammalian cells, at least sixteen polypeptides are Vagner 2009; Proudfoot 2011). dedicated to the cleavage and polyadenylation (CP) re- In mammalian cells, the pre-mRNA cleavage site is action (Chan et al. 2011; Xiang et al. 2014). Among these, determined by at least four sequence elements (Tian and cleavage and polyadenylation specificity factor (CPSF) Graber 2012): The central and most highly conserved can be considered the central complex: It carries the signal is AAUAAA or a close variant located ;20 nucle- catalytic activity for pre-mRNA cleavage, and its inter- otides (nt) upstream of the cleavage site. The preferred action with the AAUAAA sequence is essential for sequence at the cleavage site is CA. GU- or G-rich cleavage and the AAUAAA dependence of polyadenyla- downstream elements are important, and sequences up- tion. The two-subunit cleavage factor I (CF I) recognizes stream of AAUAAA, such as UGUA, can also contribute. the UGUA upstream element. CF II contains two sub- RNA sequencing (RNA-seq) experiments revealed that, in units with poorly defined functions. Cleavage stimula- many organisms, the majority of protein-coding genes tion factor (CstF) has three different subunits and have multiple polyadenylation sites generating either recognizes downstream elements. Symplekin is consid- different protein isoforms or mRNA isoforms differing in ered a scaffolding protein connecting CPSF and CstF. Poly(A) the lengths of their 39 untranslated regions (UTRs) and consequently in their interaction with RNA-binding Ó 2014 Schonemann€ et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms. Corresponding author: [email protected] xhtml). After six months, it is available under a Creative Commons Article published online ahead of print. Article and publication date are License (Attribution-NonCommercial 4.0 International), as described at online at http://www.genesdev.org/cgi/doi/10.1101/gad.250985.114. http://creativecommons.org/licenses/by-nc/4.0/. GENES & DEVELOPMENT 28:2381–2393 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/14; www.genesdev.org 2381 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Schonemann€ et al. polymerase generates the poly(A) tail and can also con- ing to an AAUAAA-containing RNA in comparison with tribute to cleavage. Although CPSF and poly(A) poly- a point mutant (Murthy and Manley 1995). CPSF30 and merase are sufficient for AAUAAA-dependent polyade- hFip1 are also RNA-binding proteins but prefer U-rich nylation, the nuclear poly(A)-binding protein 1 (PABPN1) sequences (Barabino et al. 1997; Kaufmann et al. 2004). stimulates poly(A) tail extension and is essential for the Surprisingly, a comprehensive analysis of the RNA in- synthesis of a poly(A) tail of the appropriate length. In teractions of 39 processing factors by UV cross-linking Saccharomyces cerevisiae, a slightly larger but mostly and immunoprecipitation (CLIP) followed by deep se- overlapping set of proteins has been identified as being quencing revealed that none of the putative CPSF sub- required for pre-mRNA 39 processing. Genetic confirma- units tested (CPSF160, CPSF100, CPSF73, CPSF30, and tion of the in vivo roles of these proteins in CP provides hFip1) showed a clear specificity for the AAUAAA the most persuasive evidence for similar functions of sequence. In contrast, the CLIP-derived preferences of their mammalian orthologs. A much larger set of ;80 other 39 processing factors matched those determined polypeptides has been identified by affinity purification of biochemically (Martin et al. 2012). In addition to specific a mammalian 39 processing complex and mass spectro- RNA binding, CPSF catalyzes pre-mRNA cleavage at the metric analysis (Shi et al. 2009). Some of these poly- site of poly(A) addition; CPSF73 is considered the endonu- peptides may contribute to the coupling of 39 processing clease (Mandel et al. 2006). CPSF100 has a related struc- to transcription and other processes. How many poly- ture. It has mutations in its active site but is thought to peptides are essential for the reaction remains to be contribute to the endonuclease activity of CPSF73 (Kolev determined. et al. 2008; Yang and Doublie 2011). Finally, CPSF also The subunit composition of CPSF has not been entirely recruits poly(A) polymerase to its substrates. Accordingly, clear. Purification of the factor, based on its activity in both CPSF160 and hFip1 interact with poly(A) polymerase polyadenylation assays, initially revealed four subunits: (Murthy and Manley 1995; Kaufmann et al. 2004). CPSF160, CPSF100, CPSF73, and CPSF30 (Bienroth et al. Here, we expressed and purified combinations of puta- 1991; Murthy and Manley 1992). A fifth putative subunit, tive CPSF subunits and determined that four of them— hFip1, was discovered on the basis of its homology with the CPSF160, CPSF30, hFip1, and WDR33—are necessary and yeast 39 processing factor Fip1p (Kaufmann et al. 2004). A sufficient to reconstitute, together with recombinant poly(A) sixth polypeptide, WDR33, was identified among the polymerase, AAUAAA-dependent polyadenylation. Both components of an affinity-purified 39 processing complex CPSF30 and WDR33 could be UV cross-linked to short (Shi et al. 2009) due to its similarity to the yeast 39 AAUAAA-containing RNAs, and transcriptome-wide processing factor Pfs2p (Ohnacker et al. 2000). The poly- mapping by photoactivatable ribonucleoside-enhanced adenylation activity of nuclear extract was abolished by CLIP (PAR-CLIP) showed that WDR33 contributes to immunodepletion of WDR33 and restored by the addition the recognition of the polyadenylation signal in vivo. of purified CPSF. Affinity purification of CPSF by means of Flag-tagged CPSF73 resulted in the copurification of CPSF160, CPSF100, CPSF30, hFip1, WDR33, and also Results symplekin (Shi et al. 2009). The plant ortholog of WDR33, Six subunits associate with and reconstitute CPSF the protein FY, has been genetically shown to be involved active in polyadenylation in 39 processing (Simpson et al. 2003) and is associated with other CPSF subunits (Herr et al. 2006; Hunt et al. 2008; To define the subunit composition of CPSF and facilitate Manzano et al. 2009). In S. cerevisiae, the orthologs of functional analyses, we sought to reconstitute the factor CPSF160, CPSF100, CPSF73, CPSF30, WDR33, and hFip1, by overexpression in insect cells. For this purpose, the together with poly(A) polymerase and Mpe1p, form the MultiBac system (Berger et al. 2004; Fitzgerald et al. 2006) polyadenylation factor I (PF I) (Preker et al. 1997), which is was used, which allows the simultaneous expression of part of a larger assembly (holo-CPF) (Nedea et al. 2003). The multiple polypeptides from a single virus. The success of symplekin ortholog Pta1p is not part of PF I but mediates its the reconstitution attempts was gauged
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