Structure-Function Analysis of Yeast mRNA Cap Methyltransferase and High-Copy Suppression of Conditional Mutants by AdoMet Synthase and the Ubiquitin Conjugating Enzyme Cdc34p

Corresponding author: Stewart Shuman, Molecular Biology Program, Sloan-Kettering Institute, 1275 York Ave., New York, NY 10021., s-shuman{at}ski.mskcc.org (E-mail)

Communicating editor: A. P. MITCHELL

Here we present a genetic analysis of the yeast cap-methylating enzyme Abd1p. To identify individual amino acids required for Abd1p function, we introduced alanine mutations at 35 positions of the 436-amino acid yeast protein. Two new recessive lethal mutations, F256A and Y330A, were identified. Alleles F256L and Y256L were viable, suggesting that hydrophobic residues at these positions sufficed for Abd1p function. Conservative mutations of Asp-178 established that an acidic moiety is essential at this position (i.e., D178E was viable whereas D178N was not). Phe-256, Tyr-330, and Asp-178 are conserved in all known cellular cap methyltransferases. We isolated temperature-sensitive abd1 alleles and found that abd1-ts cells display a rapid shut-off of protein synthesis upon shift to the restrictive temperature, without wholesale reduction in steady-state mRNA levels. These in vivo results are consistent with classical biochemical studies showing a requirement for the cap methyl group in cap-dependent translation. We explored the issue of how cap methylation might be regulated in vivo by conducting a genetic screen for high-copy suppressors of the ts growth defect of abd1 mutants. The identification of the yeast genes SAM2 and SAM1, which encode AdoMet synthase, as abd1 suppressors suggests that Abd1p function can be modulated by changes in the concentration of its substrate AdoMet. We also identified the ubiquitin conjugating enzyme Cdc34p as a high-copy abd1 suppressor. We show that mutations of Cdc34p that affect its ubiquitin conjugation activity or its capacity to interact with the E3-SCF complex abrogate its abd1 suppressor function. Moreover, the growth defect of abd1 mutants is exacerbated by cdc34-2. These findings suggest a novel role for Cdc34p in gene expression and engender a model whereby cap methylation or cap utilization is negatively regulated by a factor that is degraded when Cdc34p is overexpressed.

This article has been cited by other articles:

				S. Zheng and S. Shuman Structure-function analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase RNA, April 1, 2008; 14(4): 696 - 705. [Abstract] [Full Text] [PDF]

				S. Zheng, S. Shuman, and B. Schwer Sinefungin resistance of Saccharomyces cerevisiae arising from sam3 mutations that inactivate the AdoMet transporter or from increased expression of AdoMet synthase plus mRNA cap guanine-N7 methyltransferase Nucleic Acids Res., November 29, 2007; 35(20): 6895 - 6903. [Abstract] [Full Text] [PDF]

				V. H. Cowling and M. D. Cole The Myc Transactivation Domain Promotes Global Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain Independently of Direct DNA Binding Mol. Cell. Biol., March 15, 2007; 27(6): 2059 - 2073. [Abstract] [Full Text] [PDF]

				S. Hausmann, A. Ramirez, S. Schneider, B. Schwer, and S. Shuman Biochemical and genetic analysis of RNA cap guanine-N2 methyltransferases from Giardia lamblia and Schizosaccharomyces pombe Nucleic Acids Res., March 12, 2007; 35(5): 1411 - 1420. [Abstract] [Full Text] [PDF]

				S. Zheng, S. Hausmann, Q. Liu, A. Ghosh, B. Schwer, C. D. Lima, and S. Shuman Mutational Analysis of Encephalitozoon cuniculi mRNA Cap (Guanine-N7) Methyltransferase, Structure of the Enzyme Bound to Sinefungin, and Evidence That Cap Methyltransferase Is the Target of Sinefungin's Antifungal Activity J. Biol. Chem., November 24, 2006; 281(47): 35904 - 35913. [Abstract] [Full Text] [PDF]

				B. Schwer, S. Hausmann, S. Schneider, and S. Shuman Poxvirus mRNA Cap Methyltransferase: BYPASS OF THE REQUIREMENT FOR THE STIMULATORY SUBUNIT BY MUTATIONS IN THE CATALYTIC SUBUNIT AND EVIDENCE FOR INTERSUBUNIT ALLOSTERY J. Biol. Chem., July 14, 2006; 281(28): 18953 - 18960. [Abstract] [Full Text] [PDF]

				G. L. Chrebet, D. Wisniewski, A. L. Perkins, Q. Deng, M. B. Kurtz, A. Marcy, and S. A. Parent Cell-Based Assays to Detect Inhibitors of Fungal mRNA Capping Enzymes and Characterization of Sinefungin as a Cap Methyltransferase Inhibitor J Biomol Screen, June 1, 2005; 10(4): 355 - 364. [Abstract] [PDF]

				S. Hausmann, S. Zheng, C. Fabrega, S. W. Schneller, C. D. Lima, and S. Shuman Encephalitozoon cuniculi mRNA Cap (Guanine N-7) Methyltransferase: METHYL ACCEPTOR SPECIFICITY, INHIBITION BY S-ADENOSYLMETHIONINE ANALOGS, AND STRUCTURE-GUIDED MUTATIONAL ANALYSIS J. Biol. Chem., May 27, 2005; 280(21): 20404 - 20412. [Abstract] [Full Text] [PDF]

				N. Saha, S. Shuman, and B. Schwer Yeast-Based Genetic System for Functional Analysis of Poxvirus mRNA Cap Methyltransferase J. Virol., July 1, 2003; 77(13): 7300 - 7307. [Abstract] [Full Text] [PDF]

				B. Schwer, K. Lehman, N. Saha, and S. Shuman Characterization of the mRNA Capping Apparatus of Candida albicans J. Biol. Chem., January 12, 2001; 276(3): 1857 - 1864. [Abstract] [Full Text] [PDF]

				S. Hausmann, C. P. Vivares, and S. Shuman Characterization of the mRNA Capping Apparatus of the Microsporidian Parasite Encephalitozoon cuniculi J. Biol. Chem., January 4, 2002; 277(1): 96 - 103. [Abstract] [Full Text] [PDF]