*Eucaryotic translation initiation factor 6 (eIF6) and 60S ribosome biogenesis
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Eucaryotic translation initiation 6 (eIF6), a monomeric protein of about 26 kDa, binds to the 60S ribosomal subunit and prevents the association of 60S subunit with the 40S ribosomal subunit to form the 80S ribosome. Because of this ribosomal subunit anti-association property, eIF6 was thought to play a direct role in the generation of ribosomal subunits required for initiation of protein synthesis. The protein was therefore classified as a translation initiation factor, although its role in translation of mRNA has never been demonstrated.;In this thesis, we have carried out studies to understand the function of eIF6 in translation in vivo and in vitro. A 1.1 kb human cDNA that encodes eIF6 of 245 amino adds (calculated Mr 26,558) was cloned and expressed in Escherichia coli. The purified recombinant protein exhibits biochemical properties that are similar to eIF6 isolated from mammalian cell extracts. When the predicted amino add sequence of human eIF6 was used to search the yeast genomic sequence database for homologous protein in the yeast Saccharomyces cerevisiae, we identified an unknown ORF (YPRO16C) of 245 amino adds, with 72% identity to human eIF6. Cloning and expression of this ORF in E. coli yielded a recombinant protein of 245 amino adds (calculated Mr = 25,550) with ribosomal subunit anti-association activity. Characterization of this yeast gene, designated TIF6, showed that it is a single-copy gene that maps on chromosome XVI and is essential for cell growth and viability. eIF6 (Tif6p) expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. The protein was found evenly distributed throughout the cytoplasm and nucleus by fluorescence studies using GFP-tagged Tif6p and by subcellular fractionation of cells expressing c-Myc-tagged Tif6p.;To understand the role of eIF6 in protein synthesis, we made a yeast strain where Tif6p can be conditionally depleted from yeast cells. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro . These results indicate that contrary to its classification as an initiation factor, eIF6 does not function as a general translation initiation factor.;Further analysis indicated that eIF6 is not required for the stability of 60S ribosomal subunits. Rather, depletion of eIF6 from yeast cells impairs the biogenesis of 60S ribosomal subunits due to a defect in the processing of 35S pre-rRNA to 5.8S and 25S rRNAs of 60S ribosomal subunits. Database searches using mammalian eIF6 identified amino acid sequences from a number of organisms including Archeabacterium and plant, indicating that eIF6 is a evolutionary conserved protein.