A multisubunit 3' end processing factor from yeast containing poly(A) polymerase and homologues of the subunits of mammalian cleavage and polyadenylation specificity factor

Department of Cell Biology, Biozentrum, University of Basel, Switzerland.
The EMBO Journal (Impact Factor: 10.43). 09/1997; 16(15):4727-37. DOI: 10.1093/emboj/16.15.4727
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Polyadenylation is the second step in 3' end formation of most eukaryotic mRNAs. In Saccharomyces cerevisiae, this step requires three trans-acting factors: poly(A) polymerase (Pap1p), cleavage factor I (CF I) and polyadenylation factor I (PF I). Here, we describe the purification and subunit composition of a multiprotein complex containing Pap1p and PF I activities. PF I-Pap1p was purified to homogeneity by complementation of extracts mutant in the Fip1p subunit of PF I. In addition to Fip1p and Pap1p, the factor comprises homologues of all four subunits of mammalian cleavage and polyadenylation specificity factor (CPSF), as well as Ptalp, which previously has been implicated in pre-tRNA processing, and several as yet uncharacterized proteins. As expected for a PF I subunit, pta1-1 mutant extracts are deficient for polyadenylation in vitro. PF I also appears to be functionally related to CPSF, as it polyadenylates a substrate RNA more efficiently than Pap1p alone. Possibly, the observed interaction of the complex with RNA tethers Pap1p to its substrate.

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    • "The plant ortholog of WDR33, the protein FY, has been genetically shown to be involved in 39 processing (Simpson et al. 2003) and is associated with other CPSF subunits (Herr et al. 2006; Hunt et al. 2008; Manzano et al. 2009). In S. cerevisiae, the orthologs of CPSF160, CPSF100, CPSF73, CPSF30, WDR33, and hFip1, together with poly(A) polymerase and Mpe1p, form the polyadenylation factor I (PF I) (Preker et al. 1997), which is part of a larger assembly (holo-CPF) (Nedea et al. 2003). The symplekin ortholog Pta1p is not part of PF I but mediates its association with the rest of holo-CPF. "
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    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.
    Genes & Development 10/2014; 28(21). DOI:10.1101/gad.250985.114 · 10.80 Impact Factor
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    • "The protein complement of this complex machinery has been well characterized: CFI B consist of a single subunit Hrp1 [4], [5] and CF IA is comprised of the four subunits Rna14, Rna15, Pcf11 and Clp1 [6], [7], [8]. CPF consists of some fifteen subunits [9] and several sub-complexes of this factor have been characterized: Cleavage factor II (CF II) [10], which consists of Yhh1/Cft1, Pta1, Ydh1/Cft2, and the 3′ endonuclease Ysh1/Brr5 [11], [12]; polyadenylation factor I (PF I), which contains all CF II subunits, the poly(A) polymerase Pap1, Pfs1, Pfs2, Fip1 and Yth1 [13]; and the APT sub-complex in which Pti1, Ref2, Syc1, Swd2, Ssu72, Glc7 associate with PF I via the Pta1 subunit [14]. "
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    ABSTRACT: Cleavage factor IA (CF IA), cleavage and polyadenylation factor (CPF), constitute major protein complexes required for pre-mRNA 3' end formation in yeast. The Clp1 protein associates with Pcf11, Rna15 and Rna14 in CF IA but its functional role remained unclear. Clp1 carries an evolutionarily conserved P-loop motif that was previously shown to bind ATP. Interestingly, human and archaean Clp1 homologues, but not the yeast protein, carry 5' RNA kinase activity. We show that depletion of Clp1 in yeast promoted defective 3' end formation and RNA polymerase II termination; however, cells expressing Clp1 with mutant P-loops displayed only minor defects in gene expression. Similarly, purified and reconstituted mutant CF IA factors that interfered with ATP binding complemented CF IA depleted extracts in coupled in vitro transcription/3' end processing reactions. We found that Clp1 was required to assemble recombinant CF IA and that certain P-loop mutants failed to interact with the CF IA subunit Pcf11. In contrast, mutations in Clp1 enhanced binding to the 3' endonuclease Ysh1 that is a component of CPF. Our results support a structural role for the Clp1 P-loop motif. ATP binding by Clp1 likely contributes to CF IA formation and cross-factor interactions during the dynamic process of 3' end formation.
    PLoS ONE 12/2011; 6(12):e29139. DOI:10.1371/journal.pone.0029139 · 3.23 Impact Factor
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    • "CPF is thought to define the cleavage site by recognizing short U-rich tracts that flank the poly(A) site. The upstream tract is generally 10 nt or closer to the cleavage site (Preker et al. 1997; Graber et al. 1999). Structural analysis of the ternary complex of Pap1, RNA, and ATP has shown that 4 nt are needed to reach from the active site of Pap1 to the surface of the enzyme (Balbo and Bohm 2007). "
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    ABSTRACT: Synthesis of the poly(A) tail of mRNA in Saccharomyces cerevisiae requires recruitment of the polymerase Pap1 to the 3' end of cleaved pre-mRNA. This is made possible by the tethering of Pap1 to the Cleavage/Polyadenylation Factor (CPF) by Fip1. We have recently reported that Fip1 is an unstructured protein in solution, and proposed that it might maintain this conformation as part of CPF, when bound to Pap1. However, the role that this feature of Fip1 plays in 3' end processing has not been investigated. We show here that Fip1 has a flexible linker in the middle of the protein, and that removal or replacement of the linker affects the efficiency of polyadenylation. However, the point of tethering is not crucial, as a fusion protein of Pap1 and Fip1 is fully functional in cells lacking genes encoding the essential individual proteins, and directly tethering Pap1 to RNA increases the rate of poly(A) addition. We also find that the linker region of Fip1 provides a platform for critical interactions with other parts of the processing machinery. Our results indicate that the Fip1 linker, through its flexibility and protein/protein interactions, allows Pap1 to reach the 3' end of the cleaved RNA and efficiently initiate poly(A) addition.
    RNA 03/2011; 17(4):652-64. DOI:10.1261/rna.2273111 · 4.94 Impact Factor
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