Mouse CAF1 Can Function As a Processive Deadenylase/3′–5′-Exonuclease in Vitro but in Yeast the Deadenylase Function of CAF1 Is Not Required for mRNA Poly(A) Removal

The mouse CAF1 (mCAF1) is an ortholog of the yeast (y) CAF1 protein, which is a component of the CCR4-NOT complex, the major cytoplasmic deadenylase of Saccharomyces cerevisiae. Although CAF1 protein belongs to the DEDDh family of RNases, CCR4 appears to be the principle deadenylase of the CCR4-NOT complex. Here, we present evidence that mCAF1 is a processive, 3′–5′-RNase with a preference for poly(A) substrates. Like CCR4, increased length of RNA substrates converted mCAF1 into a processive enzyme. In contrast to two other DEDD family members, PAN2 and PARN, mCAF1 was not activated either by PAB1 or capped RNA substrates. The rate of deadenylation in vitro by yCCR4 and mCAF1 were both strongly influenced by secondary structures present in sequences adjacent to the poly(A) tail, suggesting that the ability of both enzymes to deadenylate might be affected by the context of the mRNA 3′-untranslated region sequences. The ability of mCAF1 to complement a ycaf1 deletion in yeast, however, did not require the RNase function of mCAF1. Importantly, yCAF1 mutations, which have been shown to block its RNase activity in vitro, did not inactivate yCAF1 in vivo, and mRNAs were deadenylated in vivo at nearly the same rate as found for wild type yCAF1. These results indicate that at least in yeast the CAF1 RNase activity is not required for its in vivo function. The mouse CAF1 (mCAF1) is an ortholog of the yeast (y) CAF1 protein, which is a component of the CCR4-NOT complex, the major cytoplasmic deadenylase of Saccharomyces cerevisiae. Although CAF1 protein belongs to the DEDDh family of RNases, CCR4 appears to be the principle deadenylase of the CCR4-NOT complex. Here, we present evidence that mCAF1 is a processive, 3′–5′-RNase with a preference for poly(A) substrates. Like CCR4, increased length of RNA substrates converted mCAF1 into a processive enzyme. In contrast to two other DEDD family members, PAN2 and PARN, mCAF1 was not activated either by PAB1 or capped RNA substrates. The rate of deadenylation in vitro by yCCR4 and mCAF1 were both strongly influenced by secondary structures present in sequences adjacent to the poly(A) tail, suggesting that the ability of both enzymes to deadenylate might be affected by the context of the mRNA 3′-untranslated region sequences. The ability of mCAF1 to complement a ycaf1 deletion in yeast, however, did not require the RNase function of mCAF1. Importantly, yCAF1 mutations, which have been shown to block its RNase activity in vitro, did not inactivate yCAF1 in vivo, and mRNAs were deadenylated in vivo at nearly the same rate as found for wild type yCAF1. These results indicate that at least in yeast the CAF1 RNase activity is not required for its in vivo function. IntroductionThe control of the rate of mRNA degradation is important to regulating the abundance and translation of mRNA in the cell. In eukaryotes a major pathway of mRNA degradation involves a poly(A)-shortening step followed by removal of the mRNA cap structure and 5′–3′ degradation of the body of the mRNA by the XRN1 exonuclease (1Tucker M. Parker R. Annu. Rev. Biochem. 2000; 69: 571-595Google Scholar). Deadenylation of the poly(A) tail is also a prerequisite to 3′–5′ degradation of the message by the exosome, most notably in control of nonsense-mediated decay (2Cao D. Parker R. Cell. 2003; 113: 533-545Google Scholar, 3Mitchell P. Tollervey D. Mol. Cell. 2003; 11: 1405-1413Google Scholar). Other analysis indicates that at least in yeast changes in the rate of mRNA deadenylation contribute to the greatest changes in the rates of mRNA degradation (4Cao D. Parker R. RNA (N. Y.). 2001; 7: 1192-1212Google Scholar). Identification of the functional deadenylases in eukaryotes and characterization of their properties is, therefore, most important to understanding the mRNA degradation process.In the yeast Saccharomyces cerevisiae the major cytoplasmic deadenylase is the CCR4-NOT complex (5Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Google Scholar, 6Tucker M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Google Scholar, 7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google Scholar, 8Daugeron M.C. Mauxion F. Seraphin B. Nucleic Acids Res. 2001; 29: 2448-2455Google Scholar). CCR4 is the principle deadenylase within this complex and is a member of the exo III family of nucleases (9Dlakic M. Trends Biochem. Sci. 2000; 25: 272-273Google Scholar). PAN2/PAN3 also functions as a deadenylase in yeast and is involved particularly in trimming of the poly(A) tail (5Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Google Scholar, 10Brown C.E. Sachs A.B. Mol. Cell. Biol. 1998; 18: 6548-6559Google Scholar). Both the CCR4-NOT complex and PAN2/PAN3 are evolutionarily conserved (11Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 3487-3495Google Scholar, 12Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Google Scholar, 13Albert T.K. Lemaire M. van Berkum N.L. Gentz R. Collart M.A. Timmers H.T. Nucleic Acids Res. 2000; 28: 809-817Google Scholar) and hCCR4 and hPAN2 display deadenylase activity in vitro (7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google Scholar, 14Uchida N. Hoshino S. Katada T. J. Biol. Chem. 2004; 279: 1383-1391Google Scholar). Higher eukaryotes also the which is a principle component of of poly(A) J. P. M. A. J. Biol. Chem. 2001; Scholar). Both PAN2 and are of the DEDD family of M.P. Nucleic Acids Res. 2001; 29: Scholar, A. Nucleic Acids Res. 25: Scholar). PAN2 is activated by the protein activated in the of the mRNA cap structure J. P. M. A. J. Biol. Chem. 2001; deadenylase CAF1 as within the CCR4-NOT complex. which CCR4 and to the of the CCR4-NOT complex (11Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 3487-3495Google Scholar, V. J. M. Denis C.L. EMBO J. 1998; is a member of the DEDDh family of M.P. Nucleic Acids Res. 2001; 29: Scholar). In vitro have that yeast CAF1 display deadenylase activity M.C. Mauxion F. Seraphin B. Nucleic Acids Res. 2001; 29: 2448-2455Google Scholar, S. Mauxion F. Seraphin B. D. EMBO 2003; the of this activity in vivo is of CAF1 not CCR4 activity in vitro (5Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Google Scholar, 7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google in the CCR4 CCR4-NOT deadenylase function in vivo M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Google Scholar, 7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google Scholar). Although a deletion the rate of in vivo poly(A) of CCR4 complement this In of CAF1 not complement a (5Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Google Scholar, H. H. H. Denis C.L. A. 1998; Scholar). of CCR4 as the major functional deadenylase activity in (7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google we have been to activity in yeast CAF1 yeast mouse the mouse CAF1 (mCAF1) protein a to the RNase in the DEDDh family of A. Nucleic Acids Res. 25: we to the deadenylase function of mCAF1. mCAF1 also been shown to complement a ycaf1 deletion in yeast M. A. T. A. A. 15: Scholar) and to strongly with yeast CCR4 and (11Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 3487-3495Google Chiang and C. suggesting that within a functional CCR4-NOT complex in the of CAF1 in mRNA we have the mCAF1 to and its in vitro and in vivo that mCAF1 is a 3′–5′ processive exonuclease with a preference for poly(A) substrates. PARN, both capped and substrates and PAN2 is by mCAF1 deadenylase activity was however, to be that of Both mCAF1 and CCR4 deadenylase function was by structures in of the poly(A) suggesting that both of RNA P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; Scholar). in either yCAF1 or mCAF1 that the in vitro function of protein the ability of either protein to complement a yeast The yCAF1 protein, deadenylation of mRNA in These results indicate that CAF1 deadenylase activity is not required for in vivo deadenylation for the other important of CAF1 in and yeast were to and In vivo deadenylation of the mRNA was in yeast with for of was with the of as Parker R. 7: Scholar). was and either or as the was a A. M. A. T. A. A. 15: Scholar). and of were and into the the were in the to of was the of A. and the RNA was as M. A. Sci. S. A. 1998; Scholar). was by with and with to the The was and of capped and RNA was with the in vitro as a The RNA was and as by the of the The RNA substrates were P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; Scholar) and of the 3′-untranslated of and with at the and of or to of and with at their and the that the as in a structure deadenylase activity of mCAF1 and and were with of mCAF1 and of CCR4 as in the to The the The of mCAF1 and CCR4 was as to rates of with secondary structure of The secondary structure for RNA was as in the to protein and were as by the with The E. were and with a The were with for at the were with or mCAF1 with or to the mCAF1 protein the The was by as (7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google vitro with RNA or in vitro RNA were with CCR4 as P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; Scholar) and with mCAF1 the was of RNase The RNA substrates were as (7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google Scholar). In vitro with RNA was as the were by analysis the of the RNA in (5Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Google Scholar) that was to the in was to the The mCAF1 were as for CCR4 P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; a have shown that CCR4 function as a deadenylase in vivo (7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google Scholar). The of CAF1 to that of of the DEDD that function as a mCAF1 function in vitro we to of in vitro RNA at its M. A. Sci. S. A. 1998; Scholar) that mCAF1 the poly(A) tail and as a into the body of the mRNA at a into the in the conserved in the exo of and the exo the exonuclease activity results with were with other substrates and with of the mCAF1 not that the activity is of mCAF1. be in with an that mCAF1 a of the or as were at this into the body of the of mCAF1 the The was not the of an as was at increased mCAF1 The to the of the structure in the RNA M. A. Sci. S. A. 1998; Scholar). These results indicate that mCAF1 is a 3′–5′ processive RNase that a preference for poly(A) analysis of mCAF1 were a and with and and and with and and with and control and and and The of the is the mCAF1 the activity with in vitro Deadenylation were as and were a at the the RNA were by a and the were with a The the or mCAF1 is not an RNA was in a mCAF1 and were at the In vitro RNA are shown the of of the mCAF1 that mCAF1 with to and with of the also a member of the DEDD family of is with capped mRNA we the activity of mCAF1 with capped and substrates. In vitro capped and were by mCAF1 at the same rates not in vitro RNA be capped in vitro, we RNA yeast and the ability of mCAF1 to the capped was by an to the of followed by with RNase to with mCAF1. of the of the RNA the cap Both capped and were with and the were by a to rates of deadenylation were for capped and as as for the that results and of the RNA in vivo Parker R. 7: Scholar). These results indicate that mCAF1 is not a be however, that present in a complex of RNA was in a by mCAF1. the poly(A) into the body of the mRNA also did not within the with a preference of mCAF1 for poly(A) substrates. also the of PAB1 mCAF1 PAB1 the ability of mCAF1 to deadenylate a and did to the same as CCR4 activity not M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Google vitro deadenylation of capped and in vivo yeast mRNA was with mCAF1 for the and the were by mCAF1 to RNA and RNA to RNA with a to the of and with RNase H. The that results in vivo and activity is at the of the The of capped and RNA with and RNase was to indicate the of the deadenylated were as with of RNA and of and of were at the the of the deadenylase activity of a RNA was P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; Scholar). at are in this RNA mCAF1 a and at the to the and to the In CCR4 at with this same P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; Scholar). the for mCAF1 with this to be and its to be The for mCAF1 was was for yeast CCR4 with a P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; the was for the of mCAF1. The of substrates were with of mCAF1 for the The were a and by a by mCAF1 and yCCR4 by in have shown P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; Scholar) that CCR4 substrates of at least in not suggesting that the RNA region was of by the enzyme. The of sequences to mCAF1 activity was by the poly(A) removal rate of mCAF1 RNA the 3′-untranslated region sequences of and The RNA was within to the and that to as were and the at and In contrast the RNA was deadenylated and in a The rate of of the was to be at least that of the rate of for the The in the of two be to sequences or secondary structures present the of and shown in secondary two structures of which of the The of mCAF1 to deadenylate the of that mCAF1 activity is strongly by rates of deadenylation activity with RNA in a this we substrates that either the of followed by the of or the of to the of The structures for two RNA are shown in RNA was to the was deadenylated to the and to two with in These to of the major structure of the RNA These results indicate two mCAF1 the rate of deadenylation of mCAF1 be influenced by sequences that within of the poly(A) sequences. mCAF1 function as a processive and within of a also the ability of CCR4 to poly(A) with same RNA substrates. RNA CCR4 and processive deadenylation was and deadenylated by CCR4 with the sequences of CCR4 activity rates are in suggesting sequences are also CCR4 deadenylation of In contrast to however, yCCR4 was to deadenylate to the suggesting be to the structure results indicate that mCAF1 not RNA a CCR4, however, this not be the structure for was in sequences of the poly(A) and CCR4 not RNA sequences. the ability of mCAF1 and CCR4 to deadenylate a structure that was within the poly(A) a the we a of the in the tail mCAF1 the structure of RNA and two and the CCR4 in by the as with deadenylated the structure with These and indicate that both mCAF1 and CCR4 deadenylation function be influenced by secondary structures not in the poly(A) region and that mCAF1 of yCAF1 and mCAF1 in both yCAF1 M.C. Mauxion F. Seraphin B. Nucleic Acids Res. 2001; 29: 2448-2455Google Scholar, S. Mauxion F. Seraphin B. D. EMBO 2003; Scholar) and mCAF1 have been shown to display deadenylase functions in vitro, we the of yCAF1 and mCAF1 were required in were either the that yCAF1 deadenylase function in vitro S. Mauxion F. Seraphin B. D. EMBO 2003; which is with the in mCAF1 that its deadenylase activity or the that is with the of mCAF1 that its The of and the of the ycaf1 deletion and not as did a not yCAF1 and mCAF1 complement of the The of yCAF1 and the of mCAF1 for the and in the of yCAF1 and mCAF1. The or of yCAF1 are in yeast (11Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 3487-3495Google Scholar). was and at V. J. M. Denis C.L. EMBO J. 1998; Scholar). (11Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 3487-3495Google Scholar) and the as were into to with the ycaf1 and were a yCAF1 a and were into the ability of to deadenylation of the mRNAs in mRNA is at two A. N. N. H. K. Nucleic Acids Res. Scholar, Denis C.L. Mol. Cell. Biol. 2003; Scholar). the of mRNA for with and at with the rate of deadenylation of the two mRNAs was followed in a ycaf1 or an deadenylation of both and mRNA and CCR4 deadenylated the two mRNAs at rates of and In in a deadenylation was for both mRNA of and deadenylation was also at in the in with that a deadenylation (5Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Google Scholar). In the deadenylated both mRNAs at rates and to as that with rate of deadenylation for was and for was These indicate that the yCAF1 deadenylase function is not required in vivo in the deadenylation also the deadenylase function of mCAF1 was required for a the of yCAF1 and the DEDDh of was for this mCAF1 M. A. T. A. A. 15: Scholar). found that mCAF1 and which mCAF1 in vitro, in yCAF1 did not with the ability of the protein to complement the of a ycaf1 deletion that inactivate yCAF1 deadenylase function in vitro not deadenylation of in and was to analysis to the rates at which mRNA deadenylated in an of the with its was at with and RNA were at the to the of and RNase to the poly(A) the of the of is with as that a member of the DEDDh family of RNases, activity in Although sequences were by its preference was for poly(A) sequences. The mCAF1 protein with substrates was a strongly processive in contrast to which to S. Mauxion F. Seraphin B. D. EMBO 2003; Scholar). RNA substrates mCAF1 not the RNA the and with substrates or the is also ability of mCAF1 to also sequences mCAF1 the CCR4, PARN, and PAN2 C.E. Sachs A.B. Mol. Cell. Biol. 1998; 18: 6548-6559Google Scholar, 14Uchida N. Hoshino S. Katada T. J. Biol. Chem. 2004; 279: 1383-1391Google Scholar, P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; Scholar, M. RNA (N. Y.). 2001; 7: Scholar, J. A.C. J. A. J. Biol. Chem. 2000; Scholar). yCAF1 also shown to with and substrates S. Mauxion F. Seraphin B. D. EMBO 2003; other in its with the two other deadenylases in the DEDD and is activated by the cap structure J. A.C. J. A. J. Biol. Chem. 2000; mCAF1 was mCAF1 was also by which is activated by PAB1 C.E. Sachs A.B. Mol. Cell. Biol. 1998; 18: 6548-6559Google Scholar, 14Uchida N. Hoshino S. Katada T. J. Biol. Chem. 2004; 279: 1383-1391Google Scholar). therefore, CCR4, a member of the exo III in its mCAF1 was in its of with or substrates mCAF1 as a processive CCR4 also the to processive with substrates in which the length was at least P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; of the length of the the of mCAF1 and CCR4 enzymes the ability of enzymes to of RNA sequences. with this was the that sequences of the poly(A) tail the deadenylase activity of both mCAF1 and CCR4 and structure in the to the poly(A) as present in the the deadenylation activity of both mCAF1 and Although we also that mCAF1 in its RNase function a the of the mCAF1 and CCR4 activity either was within a of of the These indicate that the of both enzymes be influenced by of the RNA a the of suggesting that the enzymes with a of the RNA The that the region of CCR4 is for CCR4 deadenylase activity P. G. Chiang Y.C. G. Chen J. A. Denis C.L. J. Biol. Chem. 2004; 279: Scholar) that a of the CCR4 protein be involved in its to the RNA and is of a of RNA that both mCAF1 and yCAF1 display deadenylase activity in vitro the of the of this deadenylation activity to mRNA degradation in the for mCAF1 with the for which was and found that this was that for CCR4 with substrates. Although for the yCAF1 have not been M.C. Mauxion F. Seraphin B. Nucleic Acids Res. 2001; 29: 2448-2455Google Scholar, S. Mauxion F. Seraphin B. D. EMBO 2003; an the rate of yCAF1 deadenylation S. Mauxion F. Seraphin B. D. EMBO 2003; Scholar) that yCAF1 is with Although RNA substrates mCAF1 to a processive substrates also CCR4 to the processive and of and for a of and indicate that mCAF1 is in as with These in vitro that CAF1 not contribute to the in vivo deadenylation in yCAF1 which are to yCAF1 activity in vitro S. Mauxion F. Seraphin B. D. EMBO 2003; Scholar) or are to the activity mCAF1 did not the ability of yCAF1 to complement a deletion in also that yCAF1 in deadenylation of the two mRNA at rates that were with was for wild type mCAF1 or which is in vitro, a deletion in vivo as as the wild type mCAF1 These that the deadenylation activity of yCAF1 is not required for its in vivo functions and is not for in vivo deadenylation of the we present the of CAF1 function in yeast in of mRNA CCR4 is the not functional deadenylase in the CCR4-NOT complex. deletion the rate of deadenylation in vivo and results in an to deadenylate an of (5Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Google Scholar, 6Tucker M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Google Scholar). Both of are of by of CCR4 M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Google suggesting that CAF1 a in CCR4 with the in CCR4 to the mRNA involved in or in CCR4 to other that control CCR4 with CAF1 have to with to the important CAF1 in mRNA IntroductionThe control of the rate of mRNA degradation is important to regulating the abundance and translation of mRNA in the cell. In eukaryotes a major pathway of mRNA degradation involves a poly(A)-shortening step followed by removal of the mRNA cap structure and 5′–3′ degradation of the body of the mRNA by the XRN1 exonuclease (1Tucker M. Parker R. Annu. Rev. Biochem. 2000; 69: 571-595Google Scholar). Deadenylation of the poly(A) tail is also a prerequisite to 3′–5′ degradation of the message by the exosome, most notably in control of nonsense-mediated decay (2Cao D. Parker R. Cell. 2003; 113: 533-545Google Scholar, 3Mitchell P. Tollervey D. Mol. Cell. 2003; 11: 1405-1413Google Scholar). Other analysis indicates that at least in yeast changes in the rate of mRNA deadenylation contribute to the greatest changes in the rates of mRNA degradation (4Cao D. Parker R. RNA (N. Y.). 2001; 7: 1192-1212Google Scholar). Identification of the functional deadenylases in eukaryotes and characterization of their properties is, therefore, most important to understanding the mRNA degradation process.In the yeast Saccharomyces cerevisiae the major cytoplasmic deadenylase is the CCR4-NOT complex (5Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Google Scholar, 6Tucker M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Google Scholar, 7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google Scholar, 8Daugeron M.C. Mauxion F. Seraphin B. Nucleic Acids Res. 2001; 29: 2448-2455Google Scholar). CCR4 is the principle deadenylase within this complex and is a member of the exo III family of nucleases (9Dlakic M. Trends Biochem. Sci. 2000; 25: 272-273Google Scholar). PAN2/PAN3 also functions as a deadenylase in yeast and is involved particularly in trimming of the poly(A) tail (5Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Google Scholar, 10Brown C.E. Sachs A.B. Mol. Cell. Biol. 1998; 18: 6548-6559Google Scholar). Both the CCR4-NOT complex and PAN2/PAN3 are evolutionarily conserved (11Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 3487-3495Google Scholar, 12Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Google Scholar, 13Albert T.K. Lemaire M. van Berkum N.L. Gentz R. Collart M.A. Timmers H.T. Nucleic Acids Res. 2000; 28: 809-817Google Scholar) and hCCR4 and hPAN2 display deadenylase activity in vitro (7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google Scholar, 14Uchida N. Hoshino S. Katada T. J. Biol. Chem. 2004; 279: 1383-1391Google Scholar). Higher eukaryotes also the which is a principle component of of poly(A) J. P. M. A. J. Biol. Chem. 2001; Scholar). Both PAN2 and are of the DEDD family of M.P. Nucleic Acids Res. 2001; 29: Scholar, A. Nucleic Acids Res. 25: Scholar). PAN2 is activated by the protein activated in the of the mRNA cap structure J. P. M. A. J. Biol. Chem. 2001; deadenylase CAF1 as within the CCR4-NOT complex. which CCR4 and to the of the CCR4-NOT complex (11Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 3487-3495Google Scholar, V. J. M. Denis C.L. EMBO J. 1998; is a member of the DEDDh family of M.P. Nucleic Acids Res. 2001; 29: Scholar). In vitro have that yeast CAF1 display deadenylase activity M.C. Mauxion F. Seraphin B. Nucleic Acids Res. 2001; 29: 2448-2455Google Scholar, S. Mauxion F. Seraphin B. D. EMBO 2003; the of this activity in vivo is of CAF1 not CCR4 activity in vitro (5Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Google Scholar, 7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google in the CCR4 CCR4-NOT deadenylase function in vivo M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Google Scholar, 7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google Scholar). Although a deletion the rate of in vivo poly(A) of CCR4 complement this In of CAF1 not complement a (5Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Google Scholar, H. H. H. Denis C.L. A. 1998; Scholar). of CCR4 as the major functional deadenylase activity in (7Chen J. Chiang Y.C. Denis C.L. EMBO J. 2002; 21: 1414-1426Google we have been to activity in yeast CAF1 yeast mouse the mouse CAF1 (mCAF1) protein a to the RNase in the DEDDh family of A. Nucleic Acids Res. 25: we to the deadenylase function of mCAF1. mCAF1 also been shown to complement a ycaf1 deletion in yeast M. A. T. A. A. 15: Scholar) and to strongly with yeast CCR4 and (11Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 3487-3495Google Chiang and C. suggesting that within a functional CCR4-NOT complex in the of CAF1 in mRNA we have the mCAF1 to and its in vitro and in vivo that mCAF1 is a 3′–5′ processive exonuclease with a preference for poly(A) substrates. PARN, both capped and substrates and PAN2 is by mCAF1 deadenylase activity was however, to be that of Both mCAF1 and CCR4 deadenylase function was by structures in of the poly(A) suggesting that both of RNA P. Chen J. Chiang Y.C. Denis C.L. J. Biol. Chem. 2003; Scholar). in either yCAF1 or mCAF1 that the in vitro function of protein the ability of either protein to complement a yeast The yCAF1 protein, deadenylation of mRNA in These results indicate that CAF1 deadenylase activity is not required for in vivo deadenylation for the other important of CAF1 in