CLYDE L. DENIS
Professor
Department of Molecular, Cellular and Biomedical Sciences
Program in Genetics
Ph.D., University of Washington, 1982
| |
CLYDE L. DENIS Professor Department of Molecular, Cellular and Biomedical Sciences Program in Genetics Ph.D., University of Washington, 1982 |
Our laboratory focuses on the regulation of eukaryotic mRNA production. The goal of our research is to elucidate the mechanisms that regulate mRNA deadenylation. The principal pathway for mRNA degradation in yeast involves an initial poly(A) deadenylation, followed by 5' end decapping and 5'-3' RNase digestion. The CCR4-NOT complex is the major cytoplasmic deadenylase in yeast, and is evolutionarily conserved throughout eukaryotes. CCR4 is the catalytic component of the deadenylase. A second deadenylase, PAN2/PAN3, functions in the initial trimming of the poly(A) tail.
The rate of deadenylation of different mRNA varies considerably with corresponding effects on the rate of degradation of the RNA. Moreover, the length of the poly(A) tail can alter the translatability of the mRNA. Regulation of the deadenylation process, therefore, influences multiple aspects of protein synthesis and is known to be the major factor controlling mRNA decay rates.
Two major processes have been suggested to control deadenylation. The first involves recruitment of the CCR4-NOT deadenylase to specific mRNA. Trans-acting RNA binding proteins bound to 3’ UTR sequences have been suggested in several cases to make contact to CCR4-NOT and thereby augment the rate of deadenylation. In the second case in vivo and in vitro studies have clearly indicated that the state of the mRNP structure can strongly affect the deadenylation rate. The mRNP consists of the poly(A) binding protein (PAB1) bound to the poly(A) tail, translation initiation factors (TIFs) linking the 5’ end of the mRNA to the 3’ end of the RNA, translation termination proteins, and other RNA binding proteins, such as PUF3, bound to the 3’UTR. The mechanisms by which CCR4 deadenylase activity is influenced in vivo by these mRNP factors remain largely unknown.
We have analyzed the role of the mRNP structure in regards to CCR4 deadenylation and have shown that PAB1 plays a major role in controlling deadenylation. Factors or defects in the mRNP that accelerate deadenylation have been found to do so through PAB1. For example, disrupting the TIF complex accelerates deadenylation in vivo, and we have shown that deleting either the RRM1 or P domain of PAB1 blocks this action. eIF4G of the TIF complex binds the RRM1, RRM2, and P domains of PAB1. In addition, we have found that PUF3, which accelerates deadenylation of certain mRNA, specifically requires the RRM1 domain of PAB1 for this acceleration. Also, the UPF1 protein, involved in nonsense-mediated decay, accelerates deadenylation, and we have demonstrated that it contacts PAB1 through the RRM1 domain. These observations support the model that a major part of the control of deadenylation occurs through specific contacts to PAB1.
Our current research centers on testing the model that the mRNP structure involving PAB1 plays a critical role in CCR4 deadenylation.
Click here to download Microarray Data
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.
Yao, G., Chiang, Y.-C-., Zhang, C., Lee, D., Laue, T.M., and Denis, C.L. (2007) PAB1 self-association precludes its binding to poly (A), thereby accelerating CCR4 deadenylation in vivo. Mol. Cell. Biol. 27, 6243-6253.
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.
Traven, A., Hammet, A., Tenis, N., Denis, C.L., and Heierhorst, J. (2005). Ccr4-Not complex mRNA deadenylase activity contributes to DNA damage responses in Saccharomyces cerevisiae. Genetics. 169, 65-75.
Viswanathan, P., Ohn, T., Chiang, Y.-C., Chen, J., and Denis, C.L. (2004) 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. J. Biol. Chem. 279, 23988-23995.
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. J. Biol. Chem. 279, 13616-13623.
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. Mol. Cell. Biol. 23, 7887-7901.
Denis, C.L. and Chen, J. (2003) The CCR4-NOT complex plays diverse roles in mRNA metabolism. Prog. Nucl. Acids Res. 73, 221-250.
Viswanathan, P., Chen, J., Chiang, Y.-C., and Denis, C.L. (2003) Identification of multiple RNA features that influence CCR4 deadenylation activity. J. Biol. Chem. 278, 14949-14955.
Russell, P., Benson, J.D., and Denis, C.L. (2002) Characterization of mutations in NOT2 indicates that it plays an important role in maintaining the integrity of the CCR4-NOT complex. J. Mol. Biol. 322, 27-39.
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.
Chen, J., Chiang, Y.-C., Rappsilber, J., Russell, P., Mann, M., and Denis, C.L. (2001). Purification and characterization of the 1.0MDa CCR4-NOT complex identifies two novel components of the complex. J. Mol. Biol. 314, 683-694.
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.
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.
Lui, 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.
Badarinarayana, V., Chiang, Y.-C., and Denis, C.L. (2000). Functional and physical interactions of the components of CCR4-NOT complex with TBP and its associated factors. Genetics 155, 1045-1054.
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.
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.
Komarnitsky, P.B., Klebenow, E.R., Weil, P.A., and Denis, C.L. (1998) ADR1-mediated transcriptional activational activation requires the presence of an intact TFIID complex. Mol. Cell. Biol 18, 5761-5767.
Komarnitsky, S., Chiang, Y.-C., Luca, F., Chen, J., Toyn, J., Winey, M., Johnston, L.H., and Denis, C.L. (1998). The DBF2 protein kinase binds to and acts through the cell-cycle regulated MOB1 protein. Mol. Cell. Biol. 18, 2100-2107.
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.
Hata, H., Mitsui, H., Liu, H., Bai, Y., Denis, C.L., Shimizu, Y., and Sakai, A. (1998) Dhhlp, a putative RNA helicase, associates with the general transcription factors Pop2p and Ccr4p from Saccharomyces cerevisiae. Genetics 148, 571-579.
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.