This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ho, C. K.
Right arrow Articles by Shuman, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ho, C. K.
Right arrow Articles by Shuman, S.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, September 1998, p. 5189-5198, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Genetic, Physical, and Functional Interactions between the Triphosphatase and Guanylyltransferase Components of the Yeast mRNA Capping Apparatus

C. Kiong Ho,1 Beate Schwer,2 and Stewart Shuman1,*

Molecular Biology Program, Sloan-Kettering Institute,1 and Microbiology Department, Cornell University Medical College,2 New York, New York 10021

Received 30 April 1998/Returned for modification 16 June 1998/Accepted 30 June 1998

We have characterized an essential Saccharomyces cerevisiae gene, CES5, that when present in high copy, suppresses the temperature-sensitive growth defect caused by the ceg1-25 mutation of the yeast mRNA guanylyltransferase (capping enzyme). CES5 is identical to CET1, which encodes the RNA triphosphatase component of the yeast capping apparatus. Purified recombinant Cet1 catalyzes hydrolysis of the gamma  phosphate of triphosphate-terminated RNA at a rate of 1 s-1. Cet1 is a monomer in solution; it binds with recombinant Ceg1 in vitro to form a Cet1-Ceg1 heterodimer. The interaction of Cet1 with Ceg1 elicits >10-fold stimulation of the guanylyltransferase activity of Ceg1. This stimulation is the result of increased affinity for the GTP substrate. A truncated protein, Cet1(201-549), has RNA triphosphatase activity, heterodimerizes with and stimulates Ceg1 in vitro, and suffices when expressed in single copy for cell growth in vivo. The more extensively truncated derivative Cet1(246-549) also has RNA triphosphatase activity but fails to stimulate Ceg1 in vitro and is lethal when expressed in single copy in vivo. These data suggest that the Cet1-Ceg1 interaction is essential but do not resolve whether the triphosphatase activity is also necessary. The mammalian capping enzyme Mce1 (a bifunctional triphosphatase-guanylyltransferase) substitutes for Cet1 in vivo. A mutation of the triphosphatase active-site cysteine of Mce1 is lethal. Hence, an RNA triphosphatase activity is essential for eukaryotic cell growth. This work highlights the potential for regulating mRNA cap formation through protein-protein interactions.


* Corresponding author. Mailing address: Molecular Biology Program, Sloan-Kettering Institute, 1275 York Ave., New York, NY 10021. Phone: (212) 639-7145. Fax: (212) 717-3623. E-mail: s-shuman{at}ski.mskcc.org.


Molecular and Cellular Biology, September 1998, p. 5189-5198, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.



This article has been cited by other articles:

  • Chu, C., Shatkin, A. J. (2008). Apoptosis and Autophagy Induction in Mammalian Cells by Small Interfering RNA Knockdown of mRNA Capping Enzymes. Mol. Cell. Biol. 28: 5829-5836 [Abstract] [Full Text]  
  • Takagi, Y., Sindkar, S., Ekonomidis, D., Hall, M. P., Ho, C. K. (2007). Trypanosoma brucei Encodes a Bifunctional Capping Enzyme Essential for Cap 4 Formation on the Spliced Leader RNA. J. Biol. Chem. 282: 15995-16005 [Abstract] [Full Text]  
  • Chrebet, G. L., Wisniewski, D., Perkins, A. L., Deng, Q., Kurtz, M. B., Marcy, A., Parent, S. A. (2005). Cell-Based Assays to Detect Inhibitors of Fungal mRNA Capping Enzymes and Characterization of Sinefungin as a Cap Methyltransferase Inhibitor. J Biomol Screen 10: 355-364 [Abstract]  
  • Bisaillon, M., Bougie, I. (2003). Investigating the Role of Metal Ions in the Catalytic Mechanism of the Yeast RNA Triphosphatase. J. Biol. Chem. 278: 33963-33971 [Abstract] [Full Text]  
  • Hausmann, S., Pei, Y., Shuman, S. (2003). Homodimeric Quaternary Structure Is Required for the in Vivo Function and Thermal Stability of Saccharomyces cerevisiae and Schizosaccharomyces pombe RNA Triphosphatases. J. Biol. Chem. 278: 30487-30496 [Abstract] [Full Text]  
  • Takagi, T., Walker, A. K., Sawa, C., Diehn, F., Takase, Y., Blackwell, T. K., Buratowski, S. (2003). The Caenorhabditis elegans mRNA 5'-Capping Enzyme. IN VITRO AND IN VIVO CHARACTERIZATION. J. Biol. Chem. 278: 14174-14184 [Abstract] [Full Text]  
  • Neugebauer, K. M. (2002). On the importance of being co-transcriptional. J. Cell Sci. 115: 3865-3871 [Abstract] [Full Text]  
  • Takagi, T., Cho, E.-J., Janoo, R. T. K., Polodny, V., Takase, Y., Keogh, M.-C., Woo, S.-A., Fresco-Cohen, L. D., Hoffman, C. S., Buratowski, S. (2002). Divergent Subunit Interactions among Fungal mRNA 5'-Capping Machineries. Eukaryot Cell 1: 448-457 [Abstract] [Full Text]  
  • Pei, Y., Shuman, S. (2002). Interactions between Fission Yeast mRNA Capping Enzymes and Elongation Factor Spt5. J. Biol. Chem. 277: 19639-19648 [Abstract] [Full Text]  
  • Takase, Y., Takagi, T., Komarnitsky, P. B., Buratowski, S. (2000). The Essential Interaction between Yeast mRNA Capping Enzyme Subunits Is Not Required for Triphosphatase Function In Vivo. Mol. Cell. Biol. 20: 9307-9316 [Abstract] [Full Text]  
  • Schwer, B., Saha, N., Mao, X., Chen, H.-W., Shuman, S. (2000). Structure-Function Analysis of Yeast mRNA Cap Methyltransferase and High-Copy Suppression of Conditional Mutants by AdoMet Synthase and the Ubiquitin Conjugating Enzyme Cdc34p. Genetics 155: 1561-1576 [Abstract] [Full Text]  
  • Ho, C. K., Martins, A., Shuman, S. (2000). A Yeast-Based Genetic System for Functional Analysis of Viral mRNA Capping Enzymes. J. Virol. 74: 5486-5494 [Abstract] [Full Text]  
  • Hirose, Y., Manley, J. L. (2000). RNA polymerase II and the integration of nuclear events. Genes Dev. 14: 1415-1429 [Full Text]  
  • Pei, Y., Lehman, K., Tian, L., Shuman, S. (2000). Characterization of Candida albicans RNA triphosphatase and mutational analysis of its active site. Nucleic Acids Res 28: 1885-1892 [Abstract] [Full Text]  
  • Yamada-Okabe, T., Mio, T., Kashima, Y., Matsui, M., Arisawa, M., Yamada-Okabe, H. (1999). The Candida albicans gene for mRNA 5'-cap methyltransferase: identification of additional residues essential for catalysis. Microbiology 145: 3023-3033 [Abstract] [Full Text]  
  • Pei, Y., Ho, C. K., Schwer, B., Shuman, S. (1999). Mutational Analyses of Yeast RNA Triphosphatases Highlight a Common Mechanism of Metal-dependent NTP Hydrolysis and a Means of Targeting Enzymes to Pre-mRNAs in Vivo by Fusion to the Guanylyltransferase Component of the Capping Apparatus. J. Biol. Chem. 274: 28865-28874 [Abstract] [Full Text]  
  • Lehman, K., Schwer, B., Ho, C. K., Rouzankina, I., Shuman, S. (1999). A Conserved Domain of Yeast RNA Triphosphatase Flanking the Catalytic Core Regulates Self-association and Interaction with the Guanylyltransferase Component of the mRNA Capping Apparatus. J. Biol. Chem. 274: 22668-22678 [Abstract] [Full Text]  
  • Wen, Y., Shatkin, A. J. (1999). Transcription elongation factor hSPT5 stimulates mRNA capping. Genes Dev. 13: 1774-1779 [Abstract] [Full Text]  
  • Saha, N., Schwer, B., Shuman, S. (1999). Characterization of Human, Schizosaccharomyces pombe, and Candida albicans mRNA Cap Methyltransferases and Complete Replacement of the Yeast Capping Apparatus by Mammalian Enzymes. J. Biol. Chem. 274: 16553-16562 [Abstract] [Full Text]  
  • Deshpande, T., Takagi, T., Hao, L., Buratowski, S., Charbonneau, H. (1999). Human PIR1 of the Protein-tyrosine Phosphatase Superfamily Has RNA 5'-Triphosphatase and Diphosphatase Activities. J. Biol. Chem. 274: 16590-16594 [Abstract] [Full Text]  
  • Ho, C. K., Pei, Y., Shuman, S. (1998). Yeast and Viral RNA 5' Triphosphatases Comprise a New Nucleoside Triphosphatase Family. J. Biol. Chem. 273: 34151-34156 [Abstract] [Full Text]  
  • Gross, C. H., Shuman, S. (1998). RNA 5'-Triphosphatase, Nucleoside Triphosphatase, and Guanylyltransferase Activities of Baculovirus LEF-4 Protein. J. Virol. 72: 10020-10028 [Abstract] [Full Text]  
  • Meininghaus, M., Chapman, R. D., Horndasch, M., Eick, D. (2000). Conditional Expression of RNA Polymerase II in Mammalian Cells. DELETION OF THE CARBOXYL-TERMINAL DOMAIN OF THE LARGE SUBUNIT AFFECTS EARLY STEPS IN TRANSCRIPTION. J. Biol. Chem. 275: 24375-24382 [Abstract] [Full Text]  
  • Schwer, B., Lehman, K., Saha, N., Shuman, S. (2001). Characterization of the mRNA Capping Apparatus of Candida albicans. J. Biol. Chem. 276: 1857-1864 [Abstract] [Full Text]  
  • Lehman, K., Ho, C. K., Shuman, S. (2001). Importance of Homodimerization for the in Vivo Function of Yeast RNA Triphosphatase. J. Biol. Chem. 276: 14996-15002 [Abstract] [Full Text]  
  • Bisaillon, M., Shuman, S. (2001). Structure-Function Analysis of the Active Site Tunnel of Yeast RNA Triphosphatase. J. Biol. Chem. 276: 17261-17266 [Abstract] [Full Text]  
  • Pei, Y., Hausmann, S., Ho, C. K., Schwer, B., Shuman, S. (2001). The Length, Phosphorylation State, and Primary Structure of the RNA Polymerase II Carboxyl-terminal Domain Dictate Interactions with mRNA Capping Enzymes. J. Biol. Chem. 276: 28075-28082 [Abstract] [Full Text]  
  • Bisaillon, M., Shuman, S. (2001). Functional Groups Required for the Stability of Yeast RNA Triphosphatase in Vitro and in Vivo. J. Biol. Chem. 276: 30514-30520 [Abstract] [Full Text]  
  • Hausmann, S., Ho, C. K., Schwer, B., Shuman, S. (2001). An Essential Function of Saccharomyces cerevisiae RNA Triphosphatase Cet1 Is to Stabilize RNA Guanylyltransferase Ceg1 against Thermal Inactivation. J. Biol. Chem. 276: 36116-36124 [Abstract] [Full Text]