Abstract
PUF proteins control gene expression by binding to the 3'-untranslated regions of specific mRNAs and triggering mRNA decay or translational repression. Here we focus on the mechanism of PUF-mediated regulation. The yeast PUF protein, Mpt5p, regulates HO mRNA and stimulates removal of its poly(A) tail (i.e. deadenylation). Mpt5p repression in vivo is dependent on POP2, a component of the cytoplasmic Ccr4p-Pop2p-Not complex that deadenylates mRNAs. In this study, we elucidate the individual roles of the Ccr4p and Pop2p deadenylases in Mpt5p-regulated deadenylation. Both in vivo and in vitro, Pop2p and Ccr4p proteins are required for Mpt5p-regulated deadenylation of HO. However, the requirements for the two proteins differ dramatically: the enzymatic activity of Ccr4p is essential, whereas that of Pop2p is dispensable. We conclude that Pop2p is a bridge through which the PUF protein recruits the Ccr4p enzyme to the target mRNA, thereby stimulating deadenylation. Our data suggest that PUF proteins may enhance mRNA degradation and repress expression by both deadenylation-dependent and -independent mechanisms, using the same Pop2p bridge to recruit a multifunctional Pop2p complex to the mRNA.
Highlights
The Saccharomyces cerevisiae protein Mpt5p is a member of one such family of regulatory proteins, the so-called PUF proteins [8]
The finding that Pop2p was critical for PUF-mediated regulation of HO mRNA suggested that it might act as a deadenylase on that mRNA [13]; it is controversial whether it contributes general deadenylase activity in vivo [15, 17, 19, 20, 22, 23]
CCR4 and pop2 cells carrying a wild-type copy (POP2) Are Required for Deadenylation of HO mRNA in Vivo—Mpt5p stimulates deadenylation of HO mRNA in vivo and in vitro [13]
Summary
Strains and Plasmids—The wild-type BY4742 yeast strain and isogenic strains with gene-specific deletions of POP2, CCR4, PAN2, CAF120, CAF130, NOT3, and NOT4 were obtained from Open Biosystems These deletion strains were created by PCR-mediated gene modification using the kanamycin/G418 resistance marker. Co-immunoprecipitation Analysis—Co-immunoprecipitations were performed as described by Goldstrohm et al [13], using the MPT5-TAP strain with plasmids expressing the indicated T7 epitope-tagged POP2 or CCR4 proteins. PUF Repression Growth Assays—Mpt5p-mediated repression assays were performed using the reporter gene construct YCp33 HO promoter-HIS3-HO 3Ј-UTR and YEp181 MPT5, described by Goldstrohm et al [13], in either wild-type BY4742 or pop strains. Purification of Deadenylase Complexes—Wild-type or catalytically inactive mutant Pop2p or Ccr4p complexes were purified from pop or ccr deletion strains as indicated in Fig. 4 and supplemental Fig. S1.
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