Translation Initiation in Eukaryotic Cells
The basic goal of our research is to determine the molecular mechanisms and control of initiation of protein synthesis in eukaryotic cells. There is now growing evidence that translational control along with mRNA stability and localization plays important roles in the regulation of many cellular genes. Regulation of translation occurs both temporarlly (e.g. in oogenesis, in early embryonic development, as well as in cell cycle), and spatially (e.g. synaptic long term potentiation, cell polarity and cell fate determination) as well as during signal transduction in response to mitogens and nutrients. Deregulation of translational control is also known to occur in cancer. In most translation regulatory processes studied thus far, there is compelling evidence that regulation is exerted at the level of initiation of protein synthesis.
Translation initiation is defined as the process by which ribosomes, containing bound initiator methionyl-tRNA (Met-tRNAi), are positioned at the initiation AUG codon of an mRNA to begin synthesis of a polypeptide chain. This is a very complex process that occurs by a sequence of partial reactions and requires GTP, and a large number of protein factors, called eukaryotic translation initiation factors (eIFs). In our laboratory, we use both biochemical analysis using purified mammalian initiation factors and molecular genetic approach using the budding yeast Saccharomyces cerevisiae to characterize the functions of initiation factors (eIFs). The mammalian cDNAs and the yeast genes encoding all the known initiation factors have been cloned and many of them have been isolated as functional recombinant proteins.
Currently, we are investigating the following specific problems:
(a) Previous work carried out in our laboratory has shown that following scanning of the mRNA and recognition of the AUG start codon by the 40S preinitiation complex to form the 40S initiation complex (40S•eIF3•mRNA•Met-tRNAi•eIF2•GTP), an essential initiation factor eIF5 interacts with the 40S initiation complex, and acting as a GTPase activating protein (GAP), promotes the hydrolysis of bound GTP. Hydrolysis of GTP causes the release of bound initiation factors from the 40S subunit, an event that is essential for the subsequent joining of the 60S ribosomal subunit to the 40S complex to form the elongation-competent 80S initiation complex (80S•mRNA•Met-tRNAi). Genetic evidence in the yeast S. cerevisiae indicates that eIF5-promoted GTP hydrolysis is tightly coupled to the selection of the AUG codon by the 40S preinitiation complex. This process is now being studied in vitro using purified mammalian initiation factors. The role of interaction of eIF5 with other initiation factors in coupling GTP hydrolysis to the AUG selection process will be investigated.
(b) The multi-subunit initiation factor eIF3 plays a central role in the translation initiation process. The factor is required in the initial binding of the initiator Met-tRNAi to 40S ribosomal subunits. It is also essential in the subsequent binding and scanning of the mRNA by the 40S preinitiation complex (40S•eIF3•Met-tRNAi•eIF2•GTP), leading to the recognition of the initiation AUG codon to form the 40S initiation complex. The subunit composition of eIF3 is quite complex. Mammalian eIF3 consists of five “core” subunits which are conserved in all known eukaryotes, and five “non-core” subunits which do not have structural homologues in the budding yeast, S. cerevisiae. However, the fission yeast, S. pombe encodes, in addition to the five core subunits, also four of the five non-core subunits. The in vivo function of the non-core subunits are now being analyzed using the molecular genetic techniques of S. pombe
(c) We have recently characterized both the mammalian cDNA and the yeast gene encoding the 26 kDa protein eIF6 that specifically binds to 60S ribosomal subunits. Using the molecular genetic approach in the yeast S. cerevisiae, we have shown that the protein is not involved in the translation initiation pathway. Rather, eIF6 plays an essential role in the biogenesis of 60S ribosomal subunits. Specifically, eIF6 is required for the post-transcriptional processing of pre-rRNA to mature 25S rRNA and 5.8S rRNA that are the constituents of 60S ribosomal subunits. We have also shown that eIF6 is phosphorylated in both mammalian and yeast cells at two adjacent conserved serine residues and that phosphorylation of eIF6 at these sites is required for yeast cell growth and viability. Further characterization of eIF6 particularly with regard to (a) the role of eIF6 in the export of 60S ribosomes from the nucleus to the cytoplasm and (b) the effect of phosphorylation of eIF6 in the regulation of its activity is currently being pursued.
Majumdar, R. and Maitra, U. (2005) Regulation of GTP Hydrolysis prior to Ribosomal AUG Selection during Eukaryotic Translation Initiation. EMBO J. 24, 3737-3746.
Basu, U., Si, K., Deng, H., and Maitra, U. (2003) Phosphorylation of Mammalian Eukaryotic Translation Initiation Factor 6 and its Saccharomyces cerevisiae Homologue Tif6P: Evidence that Phosphorylation of Tif6P Regulates its Nucleocytoplasmic Distribution and is Required for Yeast Cell Growth. Mol. Cell. Biol. 23, 6187-6199.
Majumdar, R., Bandyopadhyay, A., and Maitra, U. (2003). Mammalian Translation Initiation Factor eIF1 Functions with eIF1A and eIF3 in the Formation of a Stable 40 S Preinitiation Complex. J. Biol. Chem. 278, 6580-6587.
Maiti, T., Bandyopadhyay, A., and Maitra, U. (2003). Casein Kinase II Phosphorylates Translation Initiation Factor 5 (eIF5) in Saccharomyces cerevisiae. Yeast, 20, 97-108.
Bandyopadhyay, A., Viswanthan, L., Matsumoto, T., Chang, E.C. and Maitra, U. (2002) Moe1 and SpInt6, the Fission Yeast Homologues of Mammalian Translation Initiation Factor 3 Subunits p66 (eIF3d) and p48 (eIF3e), Respectively, are Required for Stable Association of eIF3 Subunits. J. Biol. Chem. 277, 2360-2367.
Majumdar, R., Bandyopadhyay, A., Deng, H. and Maitra, U. (2002) Phosphorylation of Mammalian Translation Initiation Factor 5 (eIF5) In Vitro and In Vivo. Nucleic. Acids. Res. 30, 1154-1162.
Matsumoto, S., Bandyopadhyay, A., Kwiatkowski, D.J., Maitra, U. and Matsumoto, T. (2002) Role of the Tsc1-Tsc2 Complex in Signaling and Transport Across the Cell Membrane in the Fission Yeast Schizosaccharomyces pombe. Genetics 161, 1053-1063.
Das, S. And Maitra, U. (2001) Functional Significance and Mechanism of eIF5-Promoted GTP Hydrolysis in Eukaryotic Translation Initiation. Prog. Nucleic Acid Res.Mol. Biol. 70, 207-230.
Das, S., Ghosh, R. and Maitra, U. (2001) Eukaryotic Translation Initiation Factor 5 (eIF5) Functions as a GTPase Activating Protein (GAP). J. Biol. Chem. 276, 6720-6726.
Basu, U., Si, K., Warner, J.W. and Maitra, U. (2001) The Saccharomyces cerevisiae TIF6 Gene Encoding Translation Initiation Factor 6 (eIF6) is Required for 60S Ribosomal Subunit Biogenesis. Mol. Cell. Biol. 75, 1453-1462.
Bandyopadhyay, A., Matsumoto, T. and Maitra, U. (2000) Fission yeast Int6 is not essential for global translation initiation, but deletion of int6+ causes hypersensitivity to caffeine and affects spore formation. Mol. Biol. Cell. 11, 4005-4018.
Das, S. and Maitra, U. (2000) Mutational analysis of mammalian translation Initiation Factor 5 (eIF5): role of interaction between the b-subunit of eIF2 avd eIF5 in eIF5 function in vitro. Mol. Cell. Biol. 20, 3942-3950.
Maiti, T., Das, S. and Maitra, U. (2000) Isolation and functional characterization of a temperature sensitive mutant of the yeast Saccharomyces cerevisiae in translation initiation factor eIF5: An eIF5 dependent cell-free translation system. Gene 244, 109-118.
Si, K. and Maitra, U. (1999) The Saccharomyces cerevisiae Homologue of translation initiation factor 6 does not function as a translation initiation factor. Mol. Cell. Biol. 19, 1416-1426.
Chaudhuri, J., Chowdhury, D. and Maitra, U. (1999) Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40S ribosomal preinitiation complex. J. Biol. Chem. 274, 17975-17980.
Bandyopadhyay, A., and Maitra, U. (1999) Cloning and characterization of the p42 subunit of mammalian translation initiation factor 3 (eIF3): Demonstration that eIF3 interacts with eIF5 in the mammalian cells. Nucleic Acids Res. 27, 1331-1337.