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PLOS ONE  2009 

The CCR4-NOT Complex Physically and Functionally Interacts with TRAMP and the Nuclear Exosome

DOI: 10.1371/journal.pone.0006760

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Background Ccr4-Not is a highly conserved multi-protein complex consisting in yeast of 9 subunits, including Not5 and the major yeast deadenylase Ccr4. It has been connected functionally in the nucleus to transcription by RNA polymerase II and in the cytoplasm to mRNA degradation. However, there has been no evidence so far that this complex is important for RNA degradation in the nucleus. Methodology/Principal Findings In this work we point to a new role for the Ccr4-Not complex in nuclear RNA metabolism. We determine the importance of the Ccr4-Not complex for the levels of non-coding nuclear RNAs, such as mis-processed and polyadenylated snoRNAs, whose turnover depends upon the nuclear exosome and TRAMP. Consistently, mutation of both the Ccr4-Not complex and the nuclear exosome results in synthetic slow growth phenotypes. We demonstrate physical interactions between the Ccr4-Not complex and the exosome. First, Not5 co-purifies with the exosome. Second, several exosome subunits co-purify with the Ccr4-Not complex. Third, the Ccr4-Not complex is important for the integrity of large exosome-containing complexes. Finally, we reveal a connection between the Ccr4-Not complex and TRAMP through the association of the Mtr4 helicase with the Ccr4-Not complex and the importance of specific subunits of Ccr4-Not for the association of Mtr4 with the nuclear exosome subunit Rrp6. Conclusions/Significance We propose a model in which the Ccr4-Not complex may provide a platform contributing to dynamic interactions between the nuclear exosome and its co-factor TRAMP. Our findings connect for the first time the different players involved in nuclear and cytoplasmic RNA degradation.


[1]  Butler JS (2002) The yin and yang of the exosome. Trends Cell Biol 12: 90–96.
[2]  Liu Q, Greimann JC, Lima CD (2006) Reconstitution, activities, and structure of the eukaryotic RNA exosome. Cell 127: 1223–1237.
[3]  Dziembowski A, Lorentzen E, Conti E, Seraphin B (2007) A single subunit, Dis3, is essentially responsible for yeast exosome core activity. Nat Struct Mol Biol 14: 15–22.
[4]  Allmang C, Petfalski E, Podtelejnikov A, Mann M, Tollervey D, et al. (1999) The yeast exosome and human PM-Scl are related complexes of 3′→5′ exonucleases. Genes Dev 13: 2148–2158.
[5]  Callahan KP, Butler JS (2008) Evidence for core exosome independent function of the nuclear exoribonuclease Rrp6p. Nucleic Acids Res 36: 6645–6655.
[6]  Mitchell P, Petfalski E, Houalla R, Podtelejnikov A, Mann M, et al. (2003) Rrp47p is an exosome-associated protein required for the 3′ processing of stable RNAs. Mol Cell Biol 23: 6982–6992.
[7]  Stead JA, Costello JL, Livingstone MJ, Mitchell P (2007) The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein. Nucleic Acids Res 35: 5556–5567.
[8]  Araki Y, Takahashi S, Kobayashi T, Kajiho H, Hoshino S, et al. (2001) Ski7p G protein interacts with the exosome and the Ski complex for 3′-to-5′ mRNA decay in yeast. Embo J 20: 4684–4693.
[9]  LaCava J, Houseley J, Saveanu C, Petfalski E, Thompson E, et al. (2005) RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell 121: 713–724.
[10]  Vanacova S, Wolf J, Martin G, Blank D, Dettwiler S, et al. (2005) A new yeast poly(A) polymerase complex involved in RNA quality control. PLoS Biol 3: e189.
[11]  Egecioglu DE, Henras AK, Chanfreau GF (2006) Contributions of Trf4p- and Trf5p-dependent polyadenylation to the processing and degradative functions of the yeast nuclear exosome. Rna 12: 26–32.
[12]  Houseley J, Tollervey D (2008) The nuclear RNA surveillance machinery: the link between ncRNAs and genome structure in budding yeast? Biochim Biophys Acta 1779: 239–246.
[13]  Wyers F, Rougemaille M, Badis G, Rousselle JC, Dufour ME, et al. (2005) Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell 121: 725–737.
[14]  Camblong J, Iglesias N, Fickentscher C, Dieppois G, Stutz F (2007) Antisense RNA stabilization induces transcriptional gene silencing via histone deacetylation in S. cerevisiae. Cell 131: 706–717.
[15]  Martens JA, Wu PY, Winston F (2005) Regulation of an intergenic transcript controls adjacent gene transcription in Saccharomyces cerevisiae. Genes & Dev 19: 2695–2704.
[16]  Martens JA, Laprade L, Winston F (2004) Intergenic transcription is required to repress the Saccharomyces cerevisiae SER3 gene. Nature 429: 571–574.
[17]  Hongay CF, Grisafi PL, Galitski T, Fink GR (2006) Antisense transcription controls cell fate in Saccharomyces cerevisiae. Cell 127: 735–745.
[18]  Collart MA, Timmers HT (2004) The eukaryotic Ccr4-not complex: a regulatory platform integrating mRNA metabolism with cellular signaling pathways? Prog Nucleic Acid Res Mol Biol 77: 289–322.
[19]  Collart MA (2003) Global control of gene expression in yeast by the Ccr4-Not complex. Gene 313: 1–16.
[20]  Tucker M, Valencia-Sanchez MA, Staples RR, Chen J, Denis CL, et al. (2001) The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell 104: 377–386.
[21]  Maillet L, Tu C, Hong YK, Shuster EO, Collart MA (2000) The essential function of Not1 lies within the Ccr4-Not complex. J Mol Biol 303: 131–143.
[22]  Panasenko O, Landrieux E, Feuermann M, Finka A, Paquet N, et al. (2006) The yeast Ccr4-Not complex controls ubiquitination of the nascent-associated polypeptide (NAC-EGD) complex. J Biol Chem 281: 31389–31398.
[23]  Funfschilling U, Rospert S (1999) Nascent polypeptide-associated complex stimulates protein import into yeast mitochondria. Mol Biol Cell 10: 3289–3299.
[24]  Rospert S, Dubaquié Y, Gautschi M (2002) Nascent-polypeptide-associated complex. Cell Mol Life Sci 59: 1632–1639.
[25]  Wiedmann B, Sakai H, Davis TA, Wiedmann M (1994) A protein complex required for signal-sequence-specific sorting and translocation. Nature 370: 434–440.
[26]  Panasenko OO, David FP, Collart MA (2009) Ribosome association and stability of the nascent polypeptide-associated complex is dependent upon its own ubiquitination. Genetics 181: 447–460.
[27]  Lenssen E, James N, Pedruzzi I, Dubouloz F, Cameroni E, et al. (2005) The Ccr4-Not complex independently controls both Msn2-dependent transcriptional activation–via a newly identified Glc7/Bud14 type I protein phosphatase module–and TFIID promoter distribution. Mol Cell Biol 25: 488–498.
[28]  Deluen C, James N, Maillet L, Molinete M, Theiler G, et al. (2002) The Ccr4-not complex and yTAF1 (yTaf(II)130p/yTaf(II)145p) show physical and functional interactions. Mol Cell Biol 22: 6735–6749.
[29]  Badarinarayana V, Chiang Y-C, Denis CL (2000) Functional interaction of CCR4-NOT proteins with TATAAA-binding protein (TBP) and its associated factors in yeast. Genetics 155: 1045–1054.
[30]  James N, Landrieux E, Collart MA (2007) A SAGA-independent function of SPT3 mediates transcriptional deregulation in a mutant of the Ccr4-not complex in Saccharomyces cerevisiae. Genetics 177: 123–135.
[31]  Eisenmann DM, Arndt KM, Ricupero SL, Rooney JW, Winston F (1992) SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae. Genes Dev 6: 1319–1331.
[32]  Mohibullah N, Hahn S (2008) Site-specific cross-linking of TBP in vivo and in vitro reveals a direct functional interaction with the SAGA subunit Spt3. Genes Dev 22: 2994–3006.
[33]  Westmoreland TJ, Marks JR, Olson JA Jr, Thompson EM, Resnick MA, et al. (2004) Cell cycle progression in G1 and S phases is CCR4 dependent following ionizing radiation or replication stress in Saccharomyces cerevisiae. Eukaryot Cell 3: 430–446.
[34]  Traven A, Hammet A, Tenis N, Denis CL, Heierhorst J (2005) Ccr4-not complex mRNA deadenylase activity contributes to DNA damage responses in Saccharomyces cerevisiae. Genetics 169: 65–75.
[35]  Collart MA, Struhl K (1993) CDC39, an essential nuclear protein that negatively regulates transcription and differentially affects the constitutive and inducible HIS3 promoters. Embo J 12: 177–186.
[36]  Qiu H, Hu C, Yoon S, Natarajan K, Swanson MJ, et al. (2004) An array of coactivators is required for optimal recruitment of TATA binding protein and RNA polymerase II by promoter-bound Gcn4p. Mol Cell Biol 24: 4104–4117.
[37]  Teixeira D, Parker R (2007) Analysis of P-body assembly in Saccharomyces cerevisiae. Mol Biol Cell 18: 2274–2287.
[38]  Azzouz N, Panasenko OO, Deluen C, Hsieh J, Theiler G, et al. (2009) Specific roles for the Ccr4-Not complex subunits in expression of the genome. Rna 15: 377–383.
[39]  Petfalski E, Dandekar T, Henry Y, Tollervey D (1998) Processing of the precursors to small nucleolar RNAs and rRNAs requires common components. Mol Cell Biol 18: 1181–1189.
[40]  Zagorski J, Tollervey D, Fournier MJ (1988) Characterization of an SNR gene locus in Saccharomyces cerevisiae that specifies both dispensible and essential small nuclear RNAs. Mol Cell Biol 8: 3282–3290.
[41]  van Hoof A, Lennertz P, Parker R (2000) Yeast exosome mutants accumulate 3′-extended polyadenylated forms of U4 small nuclear RNA and small nucleolar RNAs. Mol Cell Biol 20: 441–452.
[42]  Allmang C, Kufel J, Chanfreau G, Mitchell P, Petfalski E, et al. (1999) Functions of the exosome in rRNA, snoRNA and snRNA synthesis. Embo J 18: 5399–5410.
[43]  Chanfreau G, Rotondo G, Legrain P, Jacquier A (1998) Processing of a dicistronic small nucleolar RNA precursor by the RNA endonuclease Rnt1. Embo J 17: 3726–3737.
[44]  Synowsky SA, van den Heuvel RH, Mohammed S, Pijnappel PW, Heck AJ (2006) Probing genuine strong interactions and post-translational modifications in the heterogeneous yeast exosome protein complex. Mol Cell Proteomics 5: 1581–1592.
[45]  Synowsky SA, van Wijk M, Raijmakers R, Heck AJ (2008) Comparative Multiplexed Mass Spectrometric Analyses of Endogenously Expressed Yeast Nuclear and Cytoplasmic Exosomes. J Mol Biol.
[46]  Lorentzen E, Basquin J, Tomecki R, Dziembowski A, Conti E (2008) Structure of the active subunit of the yeast exosome core, Rrp44: diverse modes of substrate recruitment in the RNase II nuclease family. Mol Cell 29: 717–728.
[47]  Wang HW, Wang J, Ding F, Callahan K, Bratkowski MA, et al. (2007) Architecture of the yeast Rrp44 exosome complex suggests routes of RNA recruitment for 3′ end processing. Proc Natl Acad Sci U S A 104: 16844–16849.
[48]  Hernandez H, Dziembowski A, Taverner T, Seraphin B, Robinson CV (2006) Subunit architecture of multimeric complexes isolated directly from cells. EMBO Rep 7: 605–610.
[49]  Longtine MS, McKenzie A, Demarini DJ, Shah NG, Wach A, et al. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14: 953–961.
[50]  Jansen G, Wu C, Schade B, Thomas DY, Whiteway M (2005) Drag&Drop cloning in yeast. Gene 344: 43–51.
[51]  Lenssen E, Azzouz N, Michel A, Landrieux E, Collart MA (2007) The Ccr4-Not Complex Regulates Skn7 through Srb10 Kinase. Eukaryot Cell 6: 2251–2259.
[52]  Collart MA, Struhl K (1994) NOT1(CDC39), NOT2(CDC36), NOT3, and NOT4 encode a global-negative regulator of transcription that differentially affects TATA-element utilization. Genes Dev 8: 525–537.
[53]  Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, et al. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14: 115–132.
[54]  Thomas BJ, Rothstein R (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56: 619–630.
[55]  Zenklusen D, Vinciguerra P, Wyss JC, Stutz F (2002) Stable mRNP formation and export require cotranscriptional recruitment of the mRNA export factors Yra1p and Sub2p by Hpr1p. Mol Cell Biol 22: 8241–8253.


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