The initiation of eukaryotic protein synthesis is primarily regulated at two steps: 1) recruitment of mRNA to the 40S ribosomal subunit and 2) 40S subunit scanning to the initiation codon. However, the relative contributions that recruitment and scanning make to the overall rate of initiation are unknown. Moreover, the mechanism to explain the preferential recruitment of individual mRNAs to the 40S subunit in different states of development and growth is poorly defined. The need to understand this mechanism is particularly relevant in light of ribosome profiling data that has revealed a far greater extent to which the efficiency of mRNA recruitment is modulated in mammalian cells. Our overarching hypothesis is that the efficiencies of recruitment and scanning are modulated by the sequence and structure of the mRNA 5 untranslated region (5 UTR). Moreover, we hypothesize that changes in the phosphorylation state of initiation factors in response to environmental stimuli controls the preferential selection of mRNA to the 40S subunit. To address these gaps in our knowledge, this proposal aims to quantitatively analyze the effects of 5 UTR mRNA sequence and structure and the role of initiation factors in controlling recruitment and scanning. We will use state-of-the-art fluorescence-based assays to observe the rates of mRNA restructuring and recruitment to the human ribosome in real time. A highly purified complete reconstituted system that we have developed during the previous funding period will form the cornerstone of this proposal. Importantly, our in vitro work using purified components will be used to guide the use of cell-based experiments to better understand how mRNA structure regulates translation initiation rates. Together, the results of these experiments will provide many new insights into the molecular mechanisms underlying the regulation of mRNA translation under intrinsic and extrinsic changes in cell states.
Aim 1. Determine how 5 UTR structure affects the initial selection of mRNA by the cap- binding complex. All mRNAs in the cell compete for binding to the cap-binding complex (eIF4F). To reveal how different mRNAs are selected by eIF4F, we will use fluorescence-based assays to determine the thermodynamic and kinetic parameters of mRNA binding to eIF4F. We will determine how secondary structure in the 5 UTR regulates mRNA binding to human eIF4F and determine the roles played by eIF4A, DDX3 and other initiation factors in promoting unwinding of secondary structures in the 5 UTR.
Aim 2. Determine how initiation factors regulate mRNA recruitment to the 40S subunit and promote scanning to the initiation codon. We have generated a novel FRET assay to measure the rate of mRNA binding into the decoding site of the 40S subunit. We will use this assay together with the fluorescence-based helicase assay that we have already developed to determine how initiation factors bound either to the mRNA or to the 40S subunit promote the process of mRNA recruitment to the decoding site of the 40S subunit. We will also determine how initiation factors promote 40S subunit scanning and characterize how secondary structures regulate the rate of scanning.
Aim 3. Reveal how phosphorylation of initiation factors controls mRNA recruitment to the 40S subunit. Proteomic analysis has revealed that changes in the phosphorylation state of the cap- binding complex components (eIF4E, eIF4G, eIF4A) and associated factors such as eIF4B correlates with changes in the rate of protein synthesis. We will use the kinetic assays we have developed to study the effects of eIF4E, eIF4B and eIF4G phosphorylation on mRNA recruitment and scanning.
Proteins catalyze most of the reactions on which life depends, so it is of no surprise that there are many links between the dysregulation of protein synthesis and disease progression. An understanding of the mechanism and regulation of human protein synthesis will therefore help us better understand the progression of many diseases and provide novel drug targets.
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