Our long-term goal is to understand the mechanism and control of eukaryotic translation initiation at the molecular level. Translation initiation is the rate-limiting step of protein biosynthesis and an important target for the control of gene expression. Unregulated translation initiation is an important phenotype of malignantly transformed cells. We use yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe and mammalian cells to study the mechanism and regulation of translation initiation and how translational control is integrated into the cellular growth regulation.
Our current projects focus on the mechanism and regulation of stringent start codon recognition by ribosomes in the budding yeast S. cerevisiae. Here we use all the currently available technology, including genetics, biophysics and biochemistry, to understand the key events in the complex translation initiation reaction performed by many eukaryotic translation initiation factors. One of the foci has been eIF5, the important partner of GTP-dependent Met-tRNAi binding factor eIF2, both in its activation by guanine nucleotide exchange and its inactivation by GTP hydrolysis, which triggers factor dissociation from the ribosome initiation complex. The importance of the interaction between the two factors was recently endorsed by our discovery that an ancient eIF5-mimic protein conserved in diverse eukaryotes is a competitive inhibitor for both the positive and negative function of eIF5 during translation initiation.
We study more physiological aspects of translational regulation using fission yeast S. pombe and mammalian cells. The Warburg effect of cancer cells allows aerobic glycolysis, preventing mitochondrial breakdown and shifting carbon for anabolic reactions. We find that S. pombe cells display a similar response when starved for amino acids; they suppress mitochondrial activity and modify gene transcription program for amino acid synthesis. We hypothesize that a tumor suppressor mutation, such as int-6 altering the e-subunit of eIF3, disrupts this coordination by impairing expression of key enzymes and transcription factors and thereby changing metabolic outputs and mitochondrial activities. This would lead to cellular damage and mutations, eventually fixing glycolytic balance to aerobic glycolysis. We use human cultured cells to extend the yeast finding to human relevance.
Luna, R. E., H. Arthanari, H. Hiraishi et al. 2012. The C-terminal domain of eukaryotic initiation factor 5 promotes start codon recognition by its dynamic interplay with eIF1 and eIF2β. Cell Rep 1, 689-702.
Singh, C. R., R. Watanabe, W. Chowdhury, H. Hiraishi, M. J. Murai, Y. Yamamoto, D. Miles, Y. Ikeda, M. Asano, and K. Asano. 2012. Sequential eIF5 binding to the charged disordered segments of eIF4G and eIF2β stabilizes the 48S pre-initiation complex and promotes its shift to the initiation mode. Mol. Cell. Biol. 32, 3978-3989.
Singh, C. R., R. Watanabe, D. Zhou, M. Jennings, A. Fukao, B. Lee, Y. Ikeda, J. A. Chiorini, T. Fujiwara, R. C Wek, G. D. Pavitt, and K. Asano. 2011. Mechanisms of translational regulation by a human eIF5-mimic protein. Nucl Acids Res 39, 8314–8328
Nemoto, N., T. Udagawa, T. Ohira, L. Jiang, K. Hirota, C. R. M. Wilkinson, J. Bähler, N. Jones, K. Ohta, R. C. Wek, and K. Asano. 2010. The roles of stress-activated Sty1 and Gcn2 kinases and protooncoprotein homologue Int6/eIF3e in responses to endogenous oxidative stress during histidine starvation. J. Mol. Biol. 404, 183-201
Nemoto, N., T. Udagawa, S. Wang, C. R. Singh, E. Thorson, Z. A. Winter, T. Ohira, L. Valášek, S. J. Brown, and K. Asano. 2010. Yeast 18S rRNA is directly involved in the ribosomal response to stringent AUG selection during translation initiation. J Biol Chem 285, 32200-32212.
Watanabe, R., C. R. Singh, M. J. Murai, M. Ii, S. Fox, Y. Yamamoto, and K. Asano (2010). The eIF4G HEAT domain promotes translation re-initiation in yeast both dependent on and independent of eIF4A mRNA helicase. J Biol Chem 285, 21922-21933.
Reibarkh, M., Y. Yamamoto, C.R. Singh, F. del Rio, B. Lee, R. Luna, M. Ii, G. Wagner, and K. Asano. 2008. Eukaryotic initiation factor (eIF) 1 carries two distinct eIF5-binding faces important for multifactor assembly and AUG selection. J Biol Chem 283: 1094-1103.
Udagawa, Tsuyoshi , Naoki Nemoto, Caroline R.M. Wilkinson, Jana Narashimhan, Li Jiang, Stephan Watt, Aaron Zook, Nic Jones, Ronald C. Wek, Jürg Bähler, and Katsura Asano. 2008. Int6/eIF3e promotes general translation and Atf1 abundance to modulate Sty1 MAP kinase-dependent stress response in fission yeast. J Biol Chem 283: 22063-22075.
Asano, K. and M.S. Sachs. 2007. Translation factor control of ribosome conformation during start codon selection. Genes Dev. 21: 1280-1287.
Singh, C.R., T. Udagawa, B. Lee, S. Wassink, H. He, Y. Yamamoto, J.T. Anderson, G.D. Pavitt, and K. Asano. 2007. Change in nutritional status modulates the abundance of critical pre-initiation intermediate complexes during translation initiation in vivo. J Mol Biol 370: 315-330.
Singh, C.R. , B. Lee, T. Udagawa, S.S. Mohammad-Qureshi, Y. Yamamoto, G.D. Pavitt, and K. Asano. 2006. An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation. EMBO J. 25: 4537-4546.
Singh, C.R., C. Curtis, Y. Yamamoto, N.S. Hall, D.S. Kruse, H. He, E.M. Hannig, and K. Asano. 2005. eIF5 is critical for the integrity of scanning ribosomal preinitiation complex and accurate control of GCN4 translation. Mol. Cell. Biol. 25: 5480-5491.
Yamamoto, Y., C.R. Singh, A. Marintchev, N.S. Hall, E.M. Hannig, G. Wagner, and K. Asano. 2005. The eukaryotic initiation factor (eIF) 5 HEAT domain mediates multifactor assembly and scanning with distinct interfaces to eIF1, eIF2, eIF3 and eIF4G. Proc. Acad. Natl. Sci. USA 102: 16164-16169.
Singh, C.R., Y. Yamamoto, and K. Asano. 2004. Physical association of eukaryotic initiation factor 5 (eIF5) carboxyl terminal domain with the lysine-rich eIF2β segment strongly enhances its binding to eIF3. J. Biol. Chem. 279: 49644-49655
Singh, C.R., H. He, M. Ii, Y. Yamamoto, and K. Asano. 2004. Efficient Incorporation of Eukaryotic Initiation Factor 1 into the Multifactor Complex Is Critical for Formation of Functional Ribosomal Preinitiation Complexes in Vivo. J. Biol. Chem. 279: 31910-31920.
Hui He, T. Von Der Haar, C.R. Singh, M. Ii, B. Li, A.G. Hinnebusch, J. E.G. McCarthy, and K. Asano. 2003. The yeast eIF4G HEAT domain interacts with eIF1 and eIF5 and is involved in stringent AUG selection. Mol. Cell. Biol. 23: 5431-5445.
Asano, K., A. Shalev, L. Phan, K. Nielsen, J. Clayton, L. Valsek, T.F. Donahue, and A. G. Hinnebusch. 2001. Multiple roles for the carboxyl ternimal domain of eIF5 in translation initiation complex assembly and GTPase activiation. EMBO J. 20: 2326-2337
Akiyoshi, Y., J. Clayton, L. Phan, M. Yamamoto, A.G. Hinnebusch, Y. Watanabe, and K. Asano. 2001. Fission yeast homolog of murine Int-6 protein, encoded by mouse mammary tumor virus integration site, is associated with the conserved core subunits of eukaryotic translation initiation factor 3. J. Biol. Chem. 276: 10056-10062.
Asano, Katsura, J. Clayton, A. Shalev, and A.G. Hinnebusch. 2000. A multifactor complex of eukaryotic initiation factors eIF1, eIF2, eIF3, eIF5, and initiator tRNAMet is an important translation initiation intermediate in vivo. Genes Dev. 14: 2534-2546.
Asano, Katsura, T. Krishnamoorthy, L. Phan, G.D. Pavitt, and A.G. Hinnebusch.1999. Conserved bipartite motifs in yeast eIF5 and eIF2B epsilon, GTPase-activating and guanine-nucleotide exchange factors in translation initiation, mediate binding to their common substrate eIF2. EMBO J. 18: 1673-1688.
Asano, K. and K. Mizobuchi. 1998. Copy number control of IncIa plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site. EMBO J. 17: 5201-5213.
Asano, K., H.-P. Vornlocher, N. J. Richter-Cook, W.C. Merrick, A.G. Hinnebusch, and J.W.B. Hershey. 1997. Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits: Possible roles in RNA binding and macromolecular assembly. J. Biol. Chem. 272: 27042-27052.