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  • Book
    Kyuho Han.
    The concentration of proteins is regulated by signaling mechanisms that change mRNA levels, the rate of protein degradation or the rate of protein translation. In mammalian cells, overall protein abundance is predominantly controlled at the level of translation; therefore, understanding the translational control is crucial to understand the regulation of protein abundance, a fundamental building block of life. Translational control in metazoan is often very rapid and dynamic process: upon numerous signaling inputs such as nutrients, growth factors, and various stresses, cells quickly convey these signals through downstream signaling pathways to modulate mRNA translation either in sequence specific or nonspecific manner. Nevertheless, it has been challenging to directly monitor mRNA translation in living cells due to the lack of proper methods. In this thesis, we developed a reporter system to measure real-time changes of translation rates in individual cells by conjugating translation translational regulatory motifs to sequence encoding a fluorescent protein and a controllable destabilization domain. Application of the method showed that individual cells undergo marked fluctuations in the translation rate of mRNAs with 5' terminal oligopyrimidine (TOP) motifs that regulate the synthesis of ribosomal proteins. Furthermore, we show that small and medium reductions in amino acid levels signal through different mTOR complex 1 (mTORC1)-mediated non-4EBP and 4EBP signaling pathways, respectively, while large reductions caused an eIF2A-mediated suppression of general translation. We also investigated cell-cycle dependent translational control of TOP mRNAs using our translation reporter system in combination with live cell-cycle reporter. Cells were first aligned by cell-cycle transition points either between G0 and G1 or between G1 and S using Geminin and CDK sensors respectively and relative changes of TOP mRNA translation across the transition points were measured. This study not only showed that TOP mRNA translation were up-regulated during G1, but also showed that the TOP translation peaked in early S phase and decreased toward the late cell cycle phase. Given that TOP mRNA translation directly controls the synthesis of ribosomes and translation machineries, our finding suggested the important regulatory mechanism of cell-size control during cell-cycle progression. In summary, we developed the real-time translation reporter working at the single-cell levels, and have revealed a number of novel translational behaviors, including the fluctuation in the translation rate of TOP mRNA and the possible regulatory mechanism of cell-size control through the up-regulation of TOP mRNA translation.