Article

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: 10.75). 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.

Download full-text

Full-text

Available from: Walter Keller, Jul 06, 2015
0 Followers
 · 
69 Views
  • Source
    [Show abstract] [Hide abstract]
    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 · 12.64 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 · 4.62 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Nuclear pre-mRNA 3'-end processing is vital for the production of mature mRNA and the generation of the 3' untranslated region (UTR). However, the roles and regulation of this event in cellular development remain poorly understood. Here, we report the function of a nuclear pre-mRNA 3'-end processing pathway in synapse and axon formation in C. elegans. In a genetic enhancer screen for synaptogenesis mutants, we identified a novel polyproline-rich protein, Synaptic defective enhancer-1 (SYDN-1). Loss of function of sydn-1 causes abnormal synapse and axon development, and displays striking synergistic interactions with several genes that regulate specific aspects of synapses. SYDN-1 is required in neurons and localizes to distinct regions of the nucleus. Through a genetic suppressor screen, we found that the neuronal defects of sydn-1 mutants are suppressed by loss of function in Polyadenylation factor subunit-2 (PFS-2), a conserved WD40-repeat protein that interacts with multiple subcomplexes of the pre-mRNA 3'-end processing machinery. PFS-2 partially colocalizes with SYDN-1, and SYDN-1 influences the nuclear abundance of PFS-2. Inactivation of several members of the nuclear 3'-end processing complex suppresses sydn-1 mutants. Furthermore, lack of sydn-1 can increase the activity of 3'-end processing. Our studies provide in vivo evidence for pre-mRNA 3'-end processing in synapse and axon development and identify SYDN-1 as a negative regulator of this cellular event in neurons.
    Development 07/2010; 137(13):2237-50. DOI:10.1242/dev.049692 · 6.27 Impact Factor