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: 9.82). 09/1997; 16(15):4727-37. DOI: 10.1093/emboj/16.15.4727
Source: PubMed

ABSTRACT 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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    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 01/2011; 6(12):e29139. · 3.73 Impact Factor
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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 01/2011; 17(4):652-64. · 5.09 Impact Factor
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    ABSTRACT: The Arabidopsis thaliana ortholog of the 30-kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (CPSF30) has been implicated in the responses of plants to oxidative stress, suggesting a role for alternative polyadenylation. To better understand this, poly(A) site choice was studied in a mutant (oxt6) deficient in CPSF30 expression using a genome-scale approach. The results indicate that poly(A) site choice in a large majority of Arabidopsis genes is altered in the oxt6 mutant. A number of poly(A) sites were identified that are seen only in the wild type or oxt6 mutant. Interestingly, putative polyadenylation signals associated with sites that are seen only in the oxt6 mutant are decidedly different from the canonical plant polyadenylation signal, lacking the characteristic A-rich near-upstream element (where AAUAAA can be found); this suggests that CPSF30 functions in the handling of the near-upstream element. The sets of genes that possess sites seen only in the wild type or mutant were enriched for those involved in stress and defense responses, a result consistent with the properties of the oxt6 mutant. Taken together, these studies provide new insights into the mechanisms and consequences of CPSF30-mediated alternative polyadenylation.
    The Plant Cell 11/2012; · 9.25 Impact Factor

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