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Molecular and Cellular Biology, December 2001, p. 8346-8356, Vol. 21, No. 24
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.24.8346-8356.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Mpe1, a Zinc Knuckle Protein, Is an Essential Component of Yeast Cleavage and Polyadenylation Factor Required for the Cleavage and Polyadenylation of mRNA

Le Thuy Anh Vo,1 Michèle Minet,1 Jean-Marie Schmitter,2 François Lacroute,1 and Françoise Wyers1,*

Centre de Génétique Moléculaire, UPR A2167, CNRS, 91198 Gif sur Yvette,1 and Institut Européen de Chimie et Biologie, FRE 2247, CNRS, 33607 Pessac Cedex,2 France

Received 17 July 2001/Returned for modification 22 August 2001/Accepted 24 September 2001

In Saccharomyces cerevisiae, in vitro mRNA cleavage and polyadenylation require the poly(A) binding protein, Pab1p, and two multiprotein complexes: CFI (cleavage factor I) and CPF (cleavage and polyadenylation factor). We characterized a novel essential gene, MPE1 (YKL059c), which interacts genetically with the PCF11 gene encoding a subunit of CFI. Mpe1p is an evolutionarily conserved protein, a homolog of which is encoded by the human genome. The protein sequence contains a putative RNA-binding zinc knuckle motif. MPE1 is implicated in the choice of ACT1 mRNA polyadenylation site in vivo. Extracts from a conditional mutant, mpe1-1, or from a wild-type extract immunoneutralized for Mpe1p are defective in 3'-end processing. We used the tandem affinity purification (TAP) method on strains TAP-tagged for Mpe1p or Pfs2p to show that Mpe1p, like Pfs2p, is an integral subunit of CPF. Nevertheless a stable CPF, devoid of Mpe1p, was purified from the mpe1-1 mutant strain, showing that Mpe1p is not directly involved in the stability of this complex. Consistently, Mpe1p is also not necessary for the processive polyadenylation, nonspecific for the genuine pre-mRNA 3' end, displayed by the CPF alone. However, a reconstituted assay with purified CFI, CPF, and the recombinant Pab1p showed that Mpe1p is strictly required for the specific cleavage and polyadenylation of pre-mRNA. These results show that Mpe1p plays a crucial role in 3' end formation probably by promoting the specific link between the CFI/CPF complex and pre-mRNA.

* Corresponding author. Mailing address: Centre de Genetique Moleculaire, UPR A2167, CNRS, 91198 Gif sur Yvette, France. Phone: 33-1-69-82-31-70. Fax: 33-1-69-82-31-40. E-mail: wyers{at}cgm.cnrs-gif.fr.

Molecular and Cellular Biology, December 2001, p. 8346-8356, Vol. 21, No. 24
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.24.8346-8356.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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