Share This Article:

Identification of ebs1, lsm6 and nup159 as suppressors of spt10 effects at ADH2 in Saccharomyces cerevisiae suggests post-transcriptional defects affect mRNA synthesis

Full-Text HTML Download Download as PDF (Size:309KB) PP. 276-285
DOI: 10.4236/ajmb.2012.23029    4,202 Downloads   6,821 Views  


Suppression of the effects of an spt10 mutation on ADH2 expression is a phenotype shared by a small number of genes whose protein products are either components of the CCR4-NOT complex required for mRNA deadenylation and degradation (CCR4, CAF1, NOT4) or have been shown to interact with the complex (DBF2, SRB9, SRB10). In this work, we conducted a screen for additional suppressors of spt10 at ADH2 to identify new factors related to CCR4 function. In addition to reisolating ccr4 and caf1 alleles, three previously unidentified suppressors of spt10 were obtained: ebs1, lsm6, and nup159. These three genes are known or presumed to affect mRNA export or degradation. Mutations in EBS1, LSM6 and NUP159 not only suppressed spt10-induced ADH2 expression but also, like ccr4 and caf1 defects, reduced the ability of ADH2 to derepress. None of these defects affected the expression of CCR4-NOT complex components or the formation of the CCR4-NOT complex. The reduced ADH2 expression was also not the result of increased degradation of ADH2 mRNA, as the lsm6 and nup159 alleles, like that of a ccr4 deletion, actually slowed ADH2 degradation. Our results indicate that alterations in factors that slow mRNA degradation or affect mRNA transport may also interfere with the synthesis of mRNA and suggest an integration of such events in gene expression.

Cite this paper

Anderson, B. , May, C. and Denis, C. (2012) Identification of ebs1, lsm6 and nup159 as suppressors of spt10 effects at ADH2 in Saccharomyces cerevisiae suggests post-transcriptional defects affect mRNA synthesis. American Journal of Molecular Biology, 2, 276-285. doi: 10.4236/ajmb.2012.23029.


[1] Denis, C.L. (1984) Identification of new genes involved in the regulation of yeast alcohol dehydrogenase II. Genetics 108, 833-844.
[2] Eriksson, P.R., Mendiratta, G., McLaughlin, N.B., Wolfsberg, T.G., Marino-Ramírez, L., Pompa, T.A., Jainerin, M., Landsman, D., Shen, C.H., and Clark, D.J. (2005) Global regulation by the yeast Spt10 protein is mediated through chromatin structure and the histone upstream activating sequence elements. Mol. Cell. Biol. 25, 9127-9137.
[3] Denis, C.L. and Malvar, T. (1990) The CCR4 gene from Saccharomyces cerevisiae is required for both nonfermentative and spt-mediated gene expression. Genetics 124, 283-91.
[4] Chang, M., French-Cornay, D., Fan, H.-Y., Klein, H., Denis, C.L., and Jaehning, J.A. (1999) A complex containing RNA polymerase II, Paflp, Cdc73p, Hprlp, and Ccr4p plays a role in protein kinase C signaling. Mol. Cell. Biol 19, 1056-1067.
[5] 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 J. 16, 5289-5298.
[6] Liu, H.-Y., Badarinarayana, V., Audino, D.C., 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 J. 17, 1097-1106.
[7] McKenzie, E.A., Kent, N.A., Dowell, S.J., Moreno, F., Bird, L.E., and Mellor, J. (1993) The centromere and promoter factor, 1, CPF1, of Sacchaomyces cerevisiae modulates gene activity through a family of factors including SPT21, RPD1, (SIN3), RPD3 and CCR4. Mol Gen Genet 240, 374-86.
[8] Schild, D., (1995) Suppression of a new allele of the yeast RAD52 gene by overexpression of RAD51, mutations in srs2 and ccr4, or mating-type heterozygosity. Genetics 140, 115-27.
[9] 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. J. Mol. Biol. 314, 683-694.
[10] 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. Mol. Genet. Genomics 279, 323-337.
[11] Draper, M.P., C. Salvadore, and C.L. Denis (1995) Identification of a mouse protein whose homolog in yeast is a component of the CCR4 transcriptional regulatory complex. Mol. Cell. Biol. 15, 3487-95.
[12] Collart, M.A. and K. Struhl (1993) CDC39, an essential nuclear protein that negatively regulates transcription and differentially affects the constitutive and inducible HIS3 promoters [published erratum appears in EMBO J. 1993 12, 2990]. EMBO J. 12, 177-186.
[13] Collart, M. A., and K. Struhl, (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.
[14] Badarinarayana, V., Y.-C. Chiang, and C. L. Denis, (2000) Functional interaction of CCR4-NOT proteins with TATAA-binding protein (TBP) and its associated factors in yeast. Genetics 155, 1045-1054.
[15] Denis, C.L., Y.C. Chiang, Y. Cui, and J. Chen (2001) Genetic evidence supports a role for the yeast CCR4-NOT complex in transcriptional elongation. Genetics 158, 627-634.
[16] 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 Dev. 25, 581-593.
[17] 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 J. 21, 1414-1426.
[18] Daugeron, M.-C., Mauxion, F., and Seraphin, B., (2001) The yeast POP2 gene encodes a nuclease involved in mRNA deadenylation. Nucleic Acids Res. 29, 2448-2455.
[19] Tucker, M., R.R. Staples, M.A.Valencia-Sanchez, D. Muhlrad, and R. Parker (2002) CCR4p is the catalytic sub-unit of Ccr4p/Pop2p/Notp mRNA deadenylasecomplex in Saccharomyces cerevisiae. EMBO J. 21, 1427-1436.
[20] Tucker, M., M.A. Valencia-Sanchez, R. Staples, J. Chen, C.L. Denis, and R. Parker (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] Liu, H.-Y., Chiang, Y.-C., Pan, J., Salvadore, C., Chen, J., 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 polymerase II holoenzyme. J. Biol. Chem. 276, 7541-7548.
[22] Casamassimi, A. and C. Napoli (2007) Mediator complexes and eukaryotic transcriptional regulation: an overview. Biochim. 89, 1439-1446.
[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. Mol. Cell. Biol. 19, 6642-6651.
[24] Cui, Y., and C.L. Denis, (2003) In vivo evidence that defects in the transcriptional elongation factors RPB2, TFIIS, and SPT5 enhance upstream poly (A) site utilization. Mol. Cell Biol. 23, 7887-7901.
[25] Noble, K.N., Tran, E.J., Alcazar-Roman, A.R., Hodge, C.A., Cole, C.N., and Wente, S.R. (2011) The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1. Genes Dev. 25, 1065-1077.
[26] Bouveret, E., Rigaut, G., Shevchenko, A., Wilm, M., and Seraphin, B., (2000) A Sm-like protein complex that participates in mRNA degradation. EMBO J.19, 1661-1671.
[27] Tharun, S. and R. Parker (2001) Targeting an mRNA for decapping: displacement of translation factors and association of the Lsm1p-7p complex on deadenylated yeast mRNAs Mol. Cell, 8, 1075-1083.
[28] Zhou, J., Hidaka, K., and Futcher, B., (2000) The Est1 subunit of yeast telomerase binds the Tlc1 telomerase RNA. Mol Cell Biol 20, 1947-55.
[29] Luke, B., Azzalin, C.M., Hug, N. Deplazes, A., Peter, M., and Lingner, J. (2007) Saccharomyces cerevisiae Ebs1p is a putative ortholog of human Smg7 and promotes nonsense-mediated mRNA decay. Nucl. Acids Res. 35, 7688-7697.
[30] Ford, A.S., Guan, Q., Neeno-Eckwall, E., and Culbertson, M.R. (2006) Ebs1p, a negative regulatory of gene expression controlled by the Upf proteins in the yeast Saccharomyces cerevisiae. Eukaryot. Cell 5, 301-312.
[31] Uetz, P., Giot, L., Cagney, G., Mansfield, T.A., Judson, R.S., Knight, J.R., Lockshorn, D., Narayan, V.,Srinivasan, M., Pochart, P., Gureshi-Emili, A., Li, Y., Goodwin, B., Conover, D., Kalbfleishch, T., Vijayadamodar, G., Yang, M., Johnston, M., Field, S., and Rothberg, J.M., (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623-627.
[32] 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. Nucl. Acids Res. 35, 3002-3015.
[33] Cui, Y., Chiang, Y.-C., Viswanathan, P., Lee, D.J., and Denis, C.L. (2012) SPT5 physically interacts with CCR4 and affects mRNA degradation but does not control mRNA deadenylation. Amer. J. Mol. Biol. In Press.
[34] 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. J. Mol. Biol. 399, 562-575.
[35] Natsoulis, G., Winston, F., and Boeke, J.D. (1994). The SPT10 and SPT21 genes of Saccharomyces cerevisiae. Genetics 136, 93-105.
[36] Shen, C.-H., Leblanc, B.P., Neal, C., Akhavan, R., and Clark, D.J., (2002). Targeted histone acetylation at the yeast CUP1 promoter requires the transcriptional activator, the TATA boxes, and the putative histone acetylase encoded by SPT10. Mol. Cell. Biol. 22, 6406-6416.
[37] Orphanides, G., and Reinberg, D. (2002) A unified theory of gene expression. Cell 108,439-451.
[38] 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 Genet. 5, e1000364.
[39] Capelson, M., Doucet, C., and Hetzer, M.W. (2010) Nuclear pore complexes: guardians of the nuclear genome. Cold Spring Harbor Symp. Quant. Biol. 75, 585-598.
[40] 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.
[41] Atchison, J.D. and Rout, M.P. (2012) The yeast nuclear pore complex and transport through it. Genetics 190, 855-883.
[42] 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 Dev. 12, 357-369.
[43] Squazzo, S.L., Costa, P.J., Lindstrom,D.L., Kumer, K.E., Simic, R., Jennings, J., Link, A.J., Arndt, K.M., and Hartzog, G.A., (2002) The Paf1 complex physically and functionally associates with transcription elongation factors in vivo. EMBO 21, 764-1774.
[44] Strasser, K., S. Masuda, Mason, P., Pfannstiel, J., Oppizzi, M., Rodriguez-Navarro, S., Rondon,, A., Aguilera, A., Struhl, K., Reed, R., and E. Hurt (2002) TREX is a conserved complex coupling transcription with messenger RNA export. Nature 417, 304-307.
[45] Wada, T., Takagi, T., Yamaguchi, Y., Ferdous, A., Imai, T., Hirose, S., Sugimoto, S., Yano, K., Hartzog, G.A., Winston,F., Buratowski, S., 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 & Dev. 12, 343-356.
[46] Cook, W.J., Mosley, S., Audino, D.C., Rovelli, A., Mullaney, D., Stewart, G., and Denis, C.L.(1994) Mutations in the zinc-finger region of the yeast regulatory protein ADR1 affect both DNA binding and transcriptional activation. J. Biol. Chem. 269, 9374-9379.

comments powered by Disqus

Copyright © 2017 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.