PUF Protein-mediated Deadenylation Is Catalyzed by Ccr4p

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. 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. Regulation of mRNA stability, translation, and localization ensure that a given mRNA produces the right amount of protein at the proper time and place. These events are often controlled by elements in the 3′-untranslated region (3′-UTR) 2The abbreviations used are: UTR, untranslated region; TAP, tandem affinity tag. of the mRNA (1Gray N.K. Wickens M. Annu. Rev. Cell Dev. Biol. 1998; 14: 399-458Crossref PubMed Scopus (449) Google Scholar, 2Wickens M. Goodwin E.B. Kimble J. Strickland S. Hentze M.W. Mathews M. Translational Control. 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, NY2000: 295-370Google Scholar). mRNA stability and translational regulation are linked to cytoplasmic changes in poly(A) tail lengths (3Sachs A. Sonenberg N. Hershey J. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 447-486Google Scholar, 4Kuhn U. Wahle E. Biochim. Biophys. Acta. 2004; 1678: 67-84Crossref PubMed Scopus (259) Google Scholar). In particular, poly(A) shortening (deadenylation) is correlated with translational repression and mRNA decay (1Gray N.K. Wickens M. Annu. Rev. Cell Dev. Biol. 1998; 14: 399-458Crossref PubMed Scopus (449) Google Scholar, 2Wickens M. Goodwin E.B. Kimble J. Strickland S. Hentze M.W. Mathews M. Translational Control. 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, NY2000: 295-370Google Scholar). Specific regulatory proteins and micro-RNAs bind to 3′-UTR elements to promote poly(A) shortening and either repress translation or destroy the mRNA, or both (2Wickens M. Goodwin E.B. Kimble J. Strickland S. Hentze M.W. Mathews M. Translational Control. 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, NY2000: 295-370Google Scholar, 5Behm-Ansmant I. Rehwinkel J. Doerks T. Stark A. Bork P. Izaurralde E. Genes Dev. 2006; 20: 1885-1898Crossref PubMed Scopus (741) Google Scholar, 6Wu L. Fan J. Belasco J.G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4034-4039Crossref PubMed Scopus (919) Google Scholar, 7Giraldez A.J. Mishima Y. Rihel J. Grocock R.J. Van Dongen S. Inoue K. Enright A.J. Schier A.F. Science. 2006; 312: 75-79Crossref PubMed Scopus (1244) Google Scholar). The Saccharomyces cerevisiae protein Mpt5p is a member of one such family of regulatory proteins, the so-called PUF proteins (8Wickens M. Bernstein D.S. Kimble J. Parker R. Trends Genet. 2002; 18: 150-157Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). These proteins promote deadenylation, decay, and translational repression. Mpt5p binds to the 3′-UTR of multiple target mRNAs (9Olivas W. Parker R. EMBO J. 2000; 19: 6602-6611Crossref PubMed Scopus (218) Google Scholar, 10Seay D. Hook B. Evans K. Wickens M. RNA. 2006; 12: 1594-1600Crossref PubMed Scopus (25) Google Scholar, 11Gerber A.P. Herschlag D. Brown P.O. PLoS Biol. 2004; 2: E79Crossref PubMed Scopus (519) Google Scholar) and stimulates their deadenylation and decay. In particular, Mpt5p binds a regulatory element in the 3′-UTR of HO mRNA and causes rapid deadenylation and decay of that mRNA (12Tadauchi T. Matsumoto K. Herskowitz I. Irie K. EMBO J. 2001; 20: 552-561Crossref PubMed Scopus (122) Google Scholar, 13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). The HO endonuclease is tightly controlled at multiple levels to prevent inappropriate mating-type switching and aberrant double-stranded DNA breaks (14Haber J. Annu Rev. Genet. 1998; 32: 561-599Crossref PubMed Scopus (323) Google Scholar). The Mpt5p repressor contributes to that regulation; in its absence, aberrant switching occurs at high frequency (12Tadauchi T. Matsumoto K. Herskowitz I. Irie K. EMBO J. 2001; 20: 552-561Crossref PubMed Scopus (122) Google Scholar). Recently, using a genetic assay, we showed that repression by Mpt5p requires the POP2 gene and that PUF proteins, including Mpt5p, bind directly to Pop2p (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). Pop2p is a subunit of the major cytoplasmic deadenylase complex, the Ccr4p-Pop2p-Not complex (15Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 16Denis C.L. Chen J. Prog. Nucleic Acids Res. Mol. Biol. 2003; 73: 221-250Crossref PubMed Scopus (121) Google Scholar), thus providing a direct link between PUF proteins and the deadenylation machinery. Two of the Ccr4p-Pop2p-Not complex subunits, Pop2p and Ccr4p, bear sequence similarity to nucleases, and both proteins have been reported to possess deadenylase activity in vitro (17Tucker M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Crossref PubMed Scopus (269) Google Scholar, 18Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Crossref PubMed Scopus (203) Google Scholar, 19Daugeron M.C. Mauxion F. Seraphin B. Nucleic Acids Res. 2001; 29: 2448-2455Crossref PubMed Scopus (166) Google Scholar, 20Thore S. Mauxion F. Seraphin B. Suck D. EMBO Rep. 2003; 4: 1150-1155Crossref PubMed Scopus (97) Google Scholar). However, Ccr4p is thought to be the predominant deadenylase in yeast, at least under standard growth conditions (17Tucker M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Crossref PubMed Scopus (269) Google Scholar, 18Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Crossref PubMed Scopus (203) Google Scholar, 21Parker R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (652) Google Scholar). The finding that Pop2p was critical for PUF-mediated regulation of HO mRNA suggested that it might act as a deadenylase on that mRNA (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar); it is controversial whether it contributes general deadenylase activity in vivo (15Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 17Tucker M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Crossref PubMed Scopus (269) Google Scholar, 19Daugeron M.C. Mauxion F. Seraphin B. Nucleic Acids Res. 2001; 29: 2448-2455Crossref PubMed Scopus (166) Google Scholar, 20Thore S. Mauxion F. Seraphin B. Suck D. EMBO Rep. 2003; 4: 1150-1155Crossref PubMed Scopus (97) Google Scholar, 22Viswanathan P. Ohn T. Chiang Y.C. Chen J. Denis C.L. J. Biol. Chem. 2004; 279: 23988-23995Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 23Bianchin C. Mauxion F. Sentis S. Seraphin B. Corbo L. RNA. 2005; 11: 487-494Crossref PubMed Scopus (78) Google Scholar). The Pop2p deadenylase may be regulated (20Thore S. Mauxion F. Seraphin B. Suck D. EMBO Rep. 2003; 4: 1150-1155Crossref PubMed Scopus (97) Google Scholar, 21Parker R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (652) Google Scholar, 23Bianchin C. Mauxion F. Sentis S. Seraphin B. Corbo L. RNA. 2005; 11: 487-494Crossref PubMed Scopus (78) Google Scholar), and Mpt5p might stimulate its enzymatic activity as well as target it to specific mRNAs. The individual roles of the Pop2p and Ccr4p deadenylases in regulated mRNA decay are not understood. We sought to determine which deadenylase was responsible for PUF-stimulated deadenylation and to delineate the roles of Pop2p and Ccr4p. Our data suggest that Pop2p acts as a bridge through which the PUF protein recruits the Ccr4p enzyme to the mRNA. Additional data suggest that PUF proteins may repress expression by deadenylation-dependent and -independent mechanisms, using the same Pop2p bridge. 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. The MPT5-TAP strain (Open Biosystems) was created in the S288C strain background by integrating a C-terminal TAP tag onto the coding sequence of MPT5 using PCR-mediated gene modification with a HIS3 marker. POP2 and CCR4 expression plasmids were created in the high copy vector pACG1-NT and contained N-terminal His6 and T7 epitope tags that could be cleaved off using TEV protease. The ADH1 promoter and 3′-UTR were used for expression, and the plasmids carried the Zeocin resistance marker. The active site mutant POP2 contains two missense mutations, S188A/E190A, described by Thore et al. (20Thore S. Mauxion F. Seraphin B. Suck D. EMBO Rep. 2003; 4: 1150-1155Crossref PubMed Scopus (97) Google Scholar). The active site mutant CCR4 contains missense mutation E556A described by Chen et al. (18Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Crossref PubMed Scopus (203) Google Scholar). Both mutants were created by QuikChange (Stratagene) site-directed mutagenesis. High Resolution Northern Blotting—RNA was extracted from samples using the hot acidic phenol method. Cell pellets were resuspended in 500 ul of TENS (10 mm Tris-HCl, mm and extracted with 500 of phenol by at for by for was extracted a time with and resuspended in The decay of HO mRNA poly(A) was the time of the from at as described R. S. Mol. Cell Biol. 2003; PubMed Scopus Google Scholar). of was cleaved with and HO DNA was to the The was by and to using in a were with HO 3′-UTR or The were using a were as described by et al. (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar), using the MPT5-TAP strain with plasmids the T7 POP2 or CCR4 PUF repression were using the gene HO 3′-UTR and described by et al. (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar), in either wild-type BY4742 or or mutant POP2 were from vector from strain were and to at and the of was on with or in the of of Zeocin The was to at a of mm to of the growth of or mutant Pop2p or Ccr4p were from or deletion strains as in and to the (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar) were used that the target proteins a T7 affinity tag at the with a TEV site to of the were on or at Cell were from to of in with Zeocin were and by for in of mm Tris-HCl, mm mm and mm The was at and the was and with T7 linked to for were with of and one time with of deadenylation mm Tris-HCl, mm mm P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). proteins were from in of deadenylation using of for at were by and by deadenylation In were carried in a in deadenylation mm Tris-HCl, mm mm and with sequence HO DNA with sequence was with at the and to the at a of was to Pop2p or Ccr4p (10 were to their In we to their deadenylase activity and the amount and activity used in the in CCR4 and POP2 for of HO mRNA in stimulates deadenylation of HO mRNA in vivo and in vitro (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). We sought to determine the of Pop2p and Ccr4p to PUF-stimulated deadenylation. We the of poly(A) on HO mRNA in wild-type and in strains with deletions of of Ccr4p-Pop2p-Not We on a of and deletion We used to and samples a time to the degradation of the poly(A) was extracted from and cleaved with and a specific to a HO the poly(A) of this we high and Northern The as a In wild-type HO poly(A) a of poly(A) from to The HO poly(A) were the the well deadenylation-dependent R. Song H. Nat. Struct. Mol. Biol. 2004; 11: 121-127Crossref PubMed Scopus (652) Google Scholar). In the of poly(A) to poly(A) lengths were not the time the were to and the mRNA deadenylation In to this on mRNA the of removal of the poly(A) tail was in with In HO mRNA poly(A) deadenylation was by the complex (15Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar). In both and HO mRNA deadenylation, with decay of mRNAs in and (15Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 19Daugeron M.C. Mauxion F. Seraphin B. Nucleic Acids Res. 2001; 29: 2448-2455Crossref PubMed Scopus (166) Google Scholar). We conclude that both Pop2p and Ccr4p are required for deadenylation of HO mRNA in vivo that their roles Ccr4p is required for deadenylation. Ccr4p is to HO poly(A) in the of to in the of Pop2p deadenylation from to and is critical for removal of of HO determine whether mRNA decay were required for deadenylation of HO mRNA, we mRNA from a of mutant The same high Northern as was used to determine HO poly(A) tail lengths of CCR4 a in deadenylation the subunit of the deadenylase complex, showed on HO poly(A) tail a in the of the poly(A) was and is with a for the complex in poly(A) tail Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar). of of the Ccr4p-Pop2p-Not complex, including and on HO poly(A) tail in Ccr4p-Pop2p-Not complex were not mutants were We conclude that the deadenylase on removal of HO poly(A) and that at least proteins in the described Ccr4p-Pop2p-Not complex are not for HO mRNA deadenylation. of same not repression by Mpt5p in vivo (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). POP2 for HO in of deadenylation and repression on Pop2p suggested that its deadenylase activity might be for PUF this we whether Pop2p enzymatic activity was required for PUF-stimulated deadenylation in We HO poly(A) in a wild-type copy or mutant of POP2 on The mutation in POP2 two missense that its deadenylase activity in vitro (20Thore S. Mauxion F. Seraphin B. Suck D. EMBO Rep. 2003; 4: 1150-1155Crossref PubMed Scopus (97) Google Scholar). HO mRNA in strains was with the wild-type strain and to a mutant vector mutant the deadenylation mRNAs with between and in The wild-type POP2 gene deadenylation The missense mutant of POP2 a wild-type HO poly(A) Both wild-type and mutant Pop2p were at the same not determine whether the mutant of Pop2p with Mpt5p, we whether Pop2p with Mpt5p Both mutant and wild-type of Pop2p with Mpt5p in yeast We conclude that the POP2 protein is for deadenylation of HO mRNA in vivo its enzymatic activity is Ccr4p for HO in determine whether Ccr4p activity is required for deadenylation of HO mRNA, we a wild-type or mutant CCR4 gene on mutant and the of poly(A) on HO mRNA CCR4 deletion mutants a in deadenylation that was not by vector The wild-type of poly(A) lengths was by CCR4 missense mutation in CCR4 gene a that Ccr4p activity (18Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Crossref PubMed Scopus (203) Google Scholar), the of activity Both wild-type and mutant Ccr4p were at the same levels not and with Mpt5p We conclude that Ccr4p is the deadenylase responsible for HO mRNA deadenylation in Mpt5p by Pop2p determine whether the enzymatic activity of Pop2p was required for PUF we used a HIS3 gene the HO 3′-UTR (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). Mpt5p binds to the HO 3′-UTR and the mRNA, which be on and a of the protein Mpt5p is in wild-type the mRNA is and the on In mutant repression was by either the wild-type Pop2p or the Pop2p mutant we conclude that whereas POP2 protein is its deadenylase activity is deletion of CCR4 a on Mpt5p repression (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar), suggest that deadenylation may not be the mechanism in repression of the mRNA Pop2p for PUF-stimulated in the requirements for POP2 and CCR4 proteins, we the in vitro we that PUF-mediated deadenylation (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). In this assay, deadenylation of a PUF binding site is by Pop2p from yeast, with We used this to determine whether the enzymatic activity of Pop2p was required for PUF-stimulated deadenylation. wild-type or mutant POP2 with T7 epitope were The mutant Pop2p carried the two missense that its enzyme activity (20Thore S. Mauxion F. Seraphin B. Suck D. EMBO Rep. 2003; 4: 1150-1155Crossref PubMed Scopus (97) Google Scholar). The POP2 proteins, with were from yeast and for deadenylation using HO 3′-UTR with at their We the amount and activity of Pop2p that deadenylase activity was under the conditions used and Mpt5p deadenylation by both the wild-type Pop2p and mutant Pop2p and a and Pop2p deadenylase activity is not for PUF-stimulated deadenylation in We Pop2p from a strain to determine whether Pop2p activity in the of Ccr4p. Pop2p complex deadenylase at high this complex, Ccr4p, deadenylation activity with Mpt5p Mpt5p not Pop2p deadenylase activity and Ccr4p is critical for the deadenylase activity of the Pop2p Ccr4p for PUF-stimulated in whether the enzymatic activity of Ccr4p was for PUF-stimulated deadenylation, we T7 Ccr4p from wild-type Ccr4p deadenylation In mutant Ccr4p a Ccr4p were for deadenylation and in the of Mpt5p These data that Ccr4p is responsible for deadenylase activity of the Ccr4p-Pop2p-Not complex and for PUF-stimulated deadenylation. PUF proteins directly bind the Pop2p subunit (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar), we whether Pop2p was required for deadenylation. We Ccr4p from and its the Ccr4p from mutants and wild-type were active in deadenylation the Ccr4p complex from mutants not deadenylation with the of Ccr4p to promote deadenylation Pop2p was Pop2p is required for PUF-stimulated deadenylation in In this we to PUF proteins enhance deadenylation of target mRNAs. Mpt5p with the Ccr4p-Pop2p-Not complex through a direct with the Pop2p subunit (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). the deadenylase complex acts on mRNAs to which Mpt5p is We have that Pop2p and Ccr4p are required for PUF-mediated deadenylation, both in vivo and in However, the roles of the two proteins differ The deadenylase activity of Pop2p is for PUF repression in for deadenylation of HO mRNA in and for PUF-stimulated deadenylation in yeast which was for binding to Mpt5p, deadenylase activity in vitro with or Mpt5p we conclude that Pop2p not PUF-stimulated deadenylation. the enzyme activity of Pop2p is not the protein is for PUF-mediated deadenylation, both in vivo and in Pop2p Ccr4p from deadenylation with (17Tucker M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Crossref PubMed Scopus (269) Google Scholar, 18Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Crossref PubMed Scopus (203) Google Scholar). However, Ccr4p is not by the PUF We that Pop2p recruits the Ccr4p enzyme to the RNA. may be through a direct or could be through of the Ccr4p-Pop2p-Not complex C.L. Chen J. Prog. Nucleic Acids Res. Mol. Biol. 2003; 73: 221-250Crossref PubMed Scopus (121) Google Scholar). is that between the PUF protein and of the complex are deletion of Pop2p is to PUF-mediated deadenylation (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). We not direct between Mpt5p and of the Ccr4p-Pop2p-Not and M. deletions of of the complex not HO deadenylation or repression and 13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). The is that Pop2p is required to of Ccr4p to HO mRNA by Ccr4p is the enzyme responsible for PUF-stimulated deletion of CCR4 deadenylation in vivo and in vitro, as mutation of the Ccr4p active for the of Ccr4p in PUF-stimulated deadenylation from of HO poly(A) in a strain of both PUF proteins that HO in et 13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). MPT5 is with the the poly(A) on HO mRNA are that deletion of CCR4 The Ccr4p-Pop2p-Not complex in this PUF deletion the deadenylase is to the mRNA. poly(A) removal at a Our suggest that deadenylation is not the in which Mpt5p expression of mRNAs. The of poly(A) for translation and mRNA stability is well (1Gray N.K. Wickens M. Annu. Rev. Cell Dev. Biol. 1998; 14: 399-458Crossref PubMed Scopus (449) Google Scholar, A. Sonenberg N. Hershey J. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 447-486Google Scholar, 4Kuhn U. Wahle E. Biochim. Biophys. Acta. 2004; 1678: 67-84Crossref PubMed Scopus (259) Google Scholar). PUF repression with deadenylation and degradation of target mRNAs in a of (i.e. and in M. Bernstein D.S. Kimble J. Parker R. Trends Genet. 2002; 18: 150-157Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). Ccr4p is required for deadenylation, mutants a on repression by Mpt5p in gene repression (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). In mutants are both in deadenylation and repression. for is that Mpt5p may repress translation of deadenylation. The PUF protein, which stimulates deadenylation of target repress translation of with D. R. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). We suggest that PUF proteins recruit multiple to repress target mRNAs. Pop2p is required for PUF we suggest that it as a bridge to recruit the multifunctional Ccr4p-Pop2p-Not The complex not poly(A) its Ccr4p subunit may translational repression through is to this it with Mpt5p and a well in translation J. Parker R. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, M. U. Valencia-Sanchez M.A. Parker R. RNA. 2001; PubMed Scopus Google Scholar, H. H. H. Y. Denis C.L. Y. A. 1998; Google Scholar, A. J. Nucleic Acids Res. 2006; PubMed Scopus Google Scholar). of and by Mpt5p (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar), which with Pop2p M. U. Valencia-Sanchez M.A. Parker R. RNA. 2001; PubMed Scopus Google Scholar), the of the target mRNA. Ccr4p, and their are in regulated mRNA decay and repression by proteins and by I. Rehwinkel J. Doerks T. Stark A. Bork P. Izaurralde E. Genes Dev. 2006; 20: 1885-1898Crossref PubMed Scopus (741) Google Scholar, 17Tucker M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Crossref PubMed Scopus (269) Google Scholar, J. E. Genes Dev. 2005; 19: PubMed Scopus Google Scholar, T. S. F. Nat. Struct. Biol. 2003; PubMed Scopus Google Scholar, Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, F. RNA. 2004; PubMed Scopus Google Scholar, A. Chen Y. W. S. A. Sonenberg N. Genes Dev. 2004; 18: PubMed Scopus Google Scholar). In the roles of the two deadenylases have not been is by A. S. R. J. Chen Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar, J. RNA. 2006; 12: PubMed Scopus Google Scholar). The for in regulated deadenylation and repression is The of regulatory is a regulatory the PUF The between PUF and proteins is through (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). not PUF proteins repress in the same as the Pop2p which with Ccr4p A. A.P. W. Corbo L. T. 2001; 2: PubMed Scopus Google Scholar), is active as a deadenylase C. Mauxion F. Sentis S. Seraphin B. Corbo L. RNA. 2005; 11: 487-494Crossref PubMed Scopus (78) Google Scholar) and binds to a PUF protein (13Goldstrohm A.C. Hook B.A. Seay D.J. Wickens M. Nat. Struct. Mol. Biol. 2006; 13: 533-539Crossref PubMed Scopus (247) Google Scholar). a PUF protein may recruit two active and CCR4, to target mRNAs. be of to determine whether the requirements for the Ccr4p deadenylase and a Pop2p protein bridge are PUF proteins or are a with We the of of the Wickens The at the of was in the for this with