AJMB  Vol.2 No.1 , January 2012
SPT5 affects the rate of mRNA degradation and physically interacts with CCR4 but does not control mRNA deadenylation
Abstract: The CCR4-NOT complex has been shown to have multiple roles in mRNA metabolism, including that of transcriptional elongation, mRNA transport, and nuclear exosome function, but the primary function of CCR4 and CAF1 is in the deadenylation and degradation of cytoplasmic mRNA. As previous genetic analysis supported an interaction between SPT5, known to be involved in transcriptional elongation, and that of CCR4, the physical association of SPT5 with CCR4 was examined. A two-hybrid screen utilizing the deadenylase domain of CCR4 as a bait identified SPT5 as a potential interacting protein. SPT5 at its physiological concentration was shown to immunoprecipitate CCR4 and CAF1, and in vitro purified SPT5 specifically could bind to CAF1 and the deadenylase domain of CCR4. We additionally demonstrated that mutations in SPT5 or an spt4 deletion slowed the rate of mRNA degradation, a phenotype associated with defects in the CCR4 mRNA deadenylase complex. Yet, unlike ccr4 and caf1 deletions, spt5 and spt4 defects displayed little effect on the rate of deadenylation. They also did not affect decapping or 5' - 3' degradation of mRNA. These results suggest that the interactions between SPT5/SPT4 and the CCR4-NOT complex are probably the consequences of effects involving nuclear events and do not involve the primary role of CCR4 in mRNA deadenylation and turnover.
Cite this paper: Cui, Y. , Chiang, Y. , Viswanathan, P. , Lee, D. and Denis, C. (2012) SPT5 affects the rate of mRNA degradation and physically interacts with CCR4 but does not control mRNA deadenylation. American Journal of Molecular Biology, 2, 11-20. doi: 10.4236/ajmb.2012.21002.

[1]   Decker, C. J. and Parker, R. (1993) A turnover pathway for both stable and unstable mRNAs in yeast: Evidence for a requirement for deadenylation. Genes & Development, 7, 1632-1643. doi:10.1101/gad.7.8.1632

[2]   Muhlrad, D., Decker, C.J. and Parker, R. (1994) Deadenylation of the unstable mRNA encoded by the yeast MFA2 gene leads to decapping followed by 5'-->3' digestion of the transcript. Genes & Development, 8, 855-866. doi:10.1101/gad.8.7.855

[3]   Muhlrad, D., Decker, C.J. and Parker, R. (1995) Turnover mechanisms of the stable yeast PGK1 mRNA. Molecular Cell Biology, 15, 2145-2156.

[4]   Cao, D., and Parker, R. (2001) Computational modeling of eukaryotic mRNA turnover. RNA, 7, 1192-1212. doi:10.1017/S1355838201010330

[5]   Sachs, A.B., Sarnow, P. and Hentze, M.W. (1997) Starting at the beginning, middle, and end: Translation initiation in eukaryotes. Cell, 89, 831-838. doi:10.1016/S0092-8674(00)80268-8

[6]   Chen, J., Chiang, Y.C. and Denis, C.L. (2002) CCR4, a 3' - 5' poly(A) RNA and ssDNA exonuclease, is the catalytic component of the cytoplasmic deadenylase. EMBO Journal, 21, 1414-1426. doi:10.1093/emboj/21.6.1414

[7]   Tucker, M., Staples, R.R., Valencia-Sanchez, M.A., Muhlrad, D. and Parker, R. (2002) Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae. EMBO Journal, 21, 1427- 1436. doi:10.1093/emboj/21.6.1427

[8]   Tucker, M., Valencia-Sanchez, M.A., Staples, R., Chen, J., Denis, C.L. and Parker, R. (2001) The transcription factor associated, Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell, 104, 377-386. doi:10.1016/S0092-8674(01)00225-2

[9]   Brown, C.E. and Sachs, A.B. (1998) Poly(A) tail length control in Saccharomyces cerevisiae occurs by message- specific deadenylation. Molecular Cell Biology, 18, 6548- 6559.

[10]   Daugeron, M.C., Mauxion, F. and Seraphin, B. (2001) The yeast Pop2 gene encodes a nuclease involved in mRNA deadenylation. Nucleic Acids Research, 29, 2448-2455. doi:10.1093/nar/29.12.2448

[11]   Lee, D., Ohn, T., Chiang, Y.-C., Liu, Y., Quigley, G., Yao, G. and Denis, C.L. (2010) PUF3 acceleration of deadenylation in vivo can operate independently of CCR4 activity, possibly involving effects on the PAB1-mRNP structure. Journal of Molecular Biology, 399, 562-575. doi:10.1016/j.jmb.2010.04.034

[12]   Badarinarayana, V., Chiang, Y.-C. and Denis, C.L. (2000) Functional interaction of Ccr4-not proteins with TATAA-Binding Protein (TBP) and its associated factors in yeast. Genetics, 155, 1045-1054.

[13]   Deluen, C., James, N., Maillet, L., Molinete, M., Theiler, G., Lemaire, M., Paquet, N. and Collart, M. A. (2002) The Ccr4-not complex and yTAF1 yTaf(II)130p/yTaf(II)145p) show physical and functional interactions. Molecular Cell Biology, 22, 6735-6749. doi:10.1128/MCB.22.19.6735-6749.2002

[14]   Denis, C.L., Chiang, Y.-C., Cui, Y. and Chen, J. (2001) Genetic evidence supports a role for the yeast Ccr4-not complex in transcriptional elongation. Genetics, 158, 627-634.

[15]   Kruk, J.A., Dutta, A., Fu, J., Gilmour, D.S. and Reese, J.C. (2011) The multifunctional Ccr4-not complex directly promotes transcription elongation. Genes & Development, 25, 581-593. doi:10.1101/gad.2020911

[16]   Gaillard, H., Tous, C., Botet, J., Gonzalez-Aguilera, C., Quintero, M.J., Viladevall, L., Garcia-Rubio, M.L., Rodriguez-Gil, A., Marin, A., Anno, J., revuelta, J.L., Chavez, S. and Aguilera, A. (2009) Genome-wide analysis of factors affecting transcription elongation and DNA repair: a new role for PAF and Ccr4-not in transcription-coupled repair. PLoS Genetics, 5, e1000364. doi:10.1371/journal.pgen.1000364

[17]   Azzouz, N., Panasenko, O.O., Calau, G. and Collart, M.A. (2009) The Ccr4-not complex physically and functionally interacts with TRAMP and the nuclear exosome. PLoS One, 4, e6760. doi:10.1371/journal.pone.0006760

[18]   Derr, S.C., Azzouz, N., Fuchs, S.M., Collart, M.A., Strahl, B.D., Corbett, A.H. and Laribee, R.N. (2011) The Ccr4- not complex interacts with the mRNA export machinery. PLoS One, 6, e18302. doi:10.1371/journal.pone.0018302

[19]   Govindan, M., Meng, X., Denis, C.L., Webb, P., Baxter, J. and Walfish, P. (2009) Identification of Ccr4 and other essential thyroid hormone receptor coactivators by modified yeast synthetic genetic array analysis. Proceedings of the National Academy of Sciences USA, 106, 19854-19859.

[20]   Denis, C.L. (1984) Identification of new genes involved in the regulation of yeast alcohol dehydrogenase II. Genetics, 108, 833-844.

[21]   Liu, H.-Y., Badarinarayana, V., Audino, D., Rappsilber, J., Mann, M. and Denis, C.L. (1998) The not proteins are part of the Ccr4 transcriptional complex and affect gene expression both positively and negatively. EMBO Journal, 17, 1096-1106. doi:10.1093/emboj/17.4.1096

[22]   Sakai, A., Chibazakura, T., Shimizu, Y. and Hishinuma, F. (1992) Molecular analysis of Pop2 gene, a gene required for glucose-derepression of gene expression in Saccharomyces cerevisiae. Nucleic Acids Research, 20, 6227-6233. doi:10.1093/nar/20.23.6227

[23]   Bai, Y., Salvadore, C., Chiang, Y.-C., Collart, M., Liu, H.Y. and Denis, C.L. (1999) The Ccr4 and Caf1 proteins of the Ccr4-not complex are physically and functionally separated from not2, not4, and not5. Molecular Cell Biology, 19, 6642-6651.

[24]   Chen, J., Rappsilber, J., Chiang, Y.-C., Russell, P., Mann, M. and Denis, C.L. (2001) Purification and characterization of the 1.0 MDa Ccr4-not complex identifies two novel components of the complex. Journal of Molecular Biology, 314, 683-694. doi:10.1006/jmbi.2001.5162

[25]   Cui, Y., Ramnarain, D.B., Chiang, Y.-C., Ding, L.-H., McMahon, J.S. and Denis, C.L. (2008) Genome wide expression analysis of the Ccr4-not complex indicates that it consists of three modules with the not module controlling SAGA-responsive genes. Molecular Genetics and Genomics, 279, 323-337. doi:10.1007/s00438-007-0314-1

[26]   Liu, H.-Y., Chiang, Y.-C., Pan, J., Chen, J., Salvadore, C., Audino, D.C., Badarinarayana, V., Palaniswamy, V., Anderson, B. and Denis, C.L. (2001) Characterization of Caf4 and Caf16 reveal a functional connection between the Ccr4-not complex and a subset of SRB proteins of the RNA polymerize II holoenzyme. Journal of Biological Chemistry, 276, 7541-7548. doi:10.1074/jbc.M009112200

[27]   Maillet, L., Tu, C., Hong, Y.-K., Shuster, E.O. and Collart, M.A. (2000) The essential function of Not1 lies within the Ccr4-not complex. Journal of Molecular Biology, 303, 131-143. doi:10.1006/jmbi.2000.4131

[28]   Draper, M.P., Salvadore, C. and Denis, C.L. (1995) Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the Ccr4 transcriptional regulatory complex. Molecular Cell Biology, 15, 3487-3495.

[29]   Liu, H.-Y., Toyn, J.H., Chiang, Y.-C., Draper, M.P., Johnston, L.H. and Denis, C.L. (1997) DBF2, a cell cycle-regulated protein kinase, is physically and functionally associated with the Ccr4 transcriptional regulatory complex. EMBO Journal, 16, 5289-5298. doi:10.1093/emboj/16.17.5289

[30]   Clark, L.B., Viswanathan, P., Quigley, G., Chiang, Y.-C., McMahon, J.S., Yao, G., Chen, J., Nelsbach, A. and Denis, C.L. (2004) Systematic mutagenesis of the leucine-rich repeat (LRR) domain of Ccr4 reveals specific sites for binding to Caf1 and a separate critical role for the LRR in Ccr4 deadenylase activity. Journal of Biological Chemistry, 279, 13616-13623. doi:10.1074/jbc.M313202200

[31]   Hartzog, G.A., Wada, T., Handa, H. and Winston, F. (1998) Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae. Genes & Development, 12, 357-369. doi:10.1101/gad.12.3.357

[32]   Wada, T., Takagi, T., Yamaguguhi, Y., Ferdous, A., Imai, T., Hirose, S., Sugimoto, S., Yanu, K., Hartzog, G.A., Winston, F. and Handa H. (1998) DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes & Development, 12, 343-356. doi:10.1101/gad.12.3.343

[33]   Kim, T.-K., Ebright, R.H. and Reinberg, D. (2000) Mechanism of ATP-dependent promoter melting by transcription factor IIH. Science, 288, 1418-1421. doi:10.1126/science.288.5470.1418

[34]   Yamaguchi, Y., Takagi, T., Wada, T., Yano, K., Fuquay, A., Sugimoto, S., Hasegawa, J. and Handa, H. (1999) NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell, 97, 41-51. doi:10.1016/S0092-8674(00)80713-8

[35]   Lindstrom, D.L., Squazzo, S.L., Muster, N., Burckin, T.A., Wachter, K.C., Emigh, C.A., McCleery, J.A., Yates 3rd, J.R. and Hartzog, G.A. (2003) Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins. Molecular Cell Biology, 23, 1368-1378. doi:10.1128/MCB.23.4.1368-1378.2003

[36]   Andrulis, E.D., Werner, J., Nazarian, A., Erdjument-Bromage, H., Tempst, P. and Lis, J.T. (2002) The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila. Nature, 420, 837-841. doi:10.1038/nature01181

[37]   Draper, M.P., Liu, H.Y., Nelsbach, A.H., Mosley, S.P. and Denis, C.L. (1994) CCR4 is a glucose-regulated transcription factor whose leucine-rich repeat binds several proteins important for placing Ccr4 in its proper promoter context. Molecular Cell Biology, 14. 4522-4531.

[38]   Chiang, Y. C., Komarnitsky, P., Chase, D. and Denis, C.L. (1996) ADR1 activation domains contact the histone acetyltransferase GCN5 and the core transcriptional factor TFIIB. Journal of Biological Chemistry, 271, 32359-32365. doi:10.1074/jbc.271.50.32359

[39]   Collart, M.A. and Struhl, K. (1993) CDC39, an essential nuclear protein that negatively regulates transcription and differentially affects the constitutive and inducible HIS3 promoters. EMBO Journal, 12, 177-186.

[40]   Cook, W.J. and Denis, C.L. (1993) Identification of three genes required for the glucose-dependent transcription of the yeast transcriptional activator ADR1. Current Genetics, 23, 192-200. doi:10.1007/BF00351495

[41]   Denis, C.L., Ferguson, J. and Young, E.T. (1983) mRNA levels for the fermentative alcohol dehydrogenase of Saccharomyces cerevisiae decrease upon growth on a nonfermentable carbon source. Journal of Biological Chemistry, 258, 1165-1171.

[42]   Cui, Y. and Denis, C.L. (2003) In vivo evidence that defects in the transcriptional elongation factors RPB2, TFIIS, and SPT5 enhance upstream poly(A) site utilization. Molecular Cell Biology, 23, 7887-7901. doi:10.1128/MCB.23.21.7887-7901.2003

[43]   Yao, G., Chiang, Y.-C., Zhang, C., Lee, D.J, Laue, T.M. and Denis, C.L. (2007) PAB1 self-association precludes its binding to poly(A), thereby accelerating Ccr4 deadenylation in vivo. Molecular Cell Biology, 27, 6243-6253. doi:10.1128/MCB.00734-07

[44]   Ohn, T., Chiang, Y.-C., Lee, D.J., Yao, G., Zhang, C. and Denis, C.L. (2007) CAF1 plays an important role in mRNA deadenylation separate from its contact to Ccr4. Nucleic Acids Research, 35, 3002-3015. doi:10.1093/nar/gkm196

[45]   Beelman, C.A. and Parker, R. (1995) Degradation of mRNA in eukaryotes. Cell, 81, 179-183. doi:10.1016/0092-8674(95)90326-7

[46]   Tharun, S. and Parker, R. (1999) Analysis of mutations in the yeast mRNA decapping enzyme. Genetics, 151, 1273-1285.

[47]   Miyajima, A., Nakayama, N., Miyajima, I., Arai, N., Okayama, H. and Arai, K. (1984) Analysis of full-length cDNA clones carrying GAL1 of Saccharomyces cerevisiae: A model system for cDNA expression. Nucleic Acids Research, 12, 6397-6414. doi:10.1093/nar/12.16.6397

[48]   Couttet, P., Fromont-Racine, M., Steel, D., Pictet, R. and Grange, T. (1997) Messenger RNA deadenylylation precedes decapping in mammalian cells. Proceedings of the National Academy of Sciences USA, 94, 5628-5633. doi:10.1073/pnas.94.11.5628

[49]   Denis, C.L. and Chen, J. (2003) The Ccr4-not complex plays diverse roles in mRNA metabolism. Progress in Nucleic Acid Research & Molecular Biology, 73, 221-250. doi:10.1016/S0079-6603(03)01007-9

[50]   Pei, Y. and Shuman, S. (2002) Interactions between fission yeast mRNA capping enzymes and elongation factor Spt5. Journal of Biological Chemistry, 277, 19639-19648. doi:10.1074/jbc.M200015200