title: Mpe1, a zinc knuckle protein, is an essential component of yeast cleavage and polyadenylation factor required for the cleavage and polyadenylation of mRNA.
abstract: 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.
authors.affiliation: Centre de Génétique Moléculaire, UPR A2167, CNRS, 91198 Gif sur Yvette, France.
authors.forename: L T
authors.initials: LT
authors.lastname: Vo
authors.forename: M
authors.initials: M
authors.lastname: Minet
authors.forename: J M
authors.initials: JM
authors.lastname: Schmitter
authors.forename: F
authors.initials: F
authors.lastname: Lacroute
authors.forename: F
authors.initials: F
authors.lastname: Wyers
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D015152:Blotting, Northern; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D015151:Immunoblotting; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D026723:Polyadenylation; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors; D015032:Zinc / Q000737:chemistry; D039221:mRNA Cleavage and Polyadenylation Factors
chemical_list: C441449:Mpe1 protein, S cerevisiae; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D012313:RNA; D015032:Zinc