Controlling gene expression at the level of translation allows cells to respond rapidly and precisely to internal and external changes, and defects in this process can have dire consequences. Cancer, heart disease, and other developmental and neurological disorders have been linked to defects in phosphorylation cascades that are responsible for modifying eukaryotic translation initiation factors (eIFs) and actuating global changes in translation efficiency and in the translation of specific mRNAs, yet the molecular outcome of these phosphorylation events is not understood in most cases. The long-term goal of our work is to understand the molecular basis for translational control at the level of mRNA recruitment to the ribosomal translation preinitiation complex (PIC). By further investigating how phosphorylation of eIFs changes the efficiency of translation, we will make insights into the basic molecular mechanisms underlying the etiology of cancer and other diseases. During translation initiation, eIFs and other factors interact within the PIC and elements of mRNAs to determine what mRNAs are translated. We have reconstituted translation initiation in vitro from purified yeast components and made important findings regarding the roles of eIF4B and eIF4F in promoting the mRNA recruitment step. We demonstrated that eIF4B binds directly to the ribosome, and promotes functional interaction of eIF4F with the initiation machinery to promote mRNA recruitment to the PIC, challenging the previously held view that eIF4B acts as an RNA-binding protein to activate mRNAs for translation. We also have evidence that eIF4G interacts with the PIC in an mRNA-independent manner. This leads us to hypothesize that rather than activating mRNPs independently of the ribosome as has been suggested, the eIF4 factors bind to the PIC to form a holoPIC that directly recruits and unwinds mRNA. To investigate this possibility, and to understand the basic system in place for recruiting mRNAs, we will characterize the interactions of the eIF4 factors with the PIC. We will then ask what happens to these interactions and assess the resulting changes in translation and phenotype when the eIF4 factors are phosphorylated. In the future this will allow me to ask additional important questions about the mRNAs that are translated in response to single phosphorylation events and how the general translation initiation machinery is modified to allow translational control of specific mRNAs, distinguishing me from my mentor, Jon Lorsch. In the course of pursuing these questions, I will gain new skills and set up an exciting project that will make me a more competitive candidate for faculty positions. The award would provide the resources to help me achieve my goal of becoming an independent researcher, and allow me to acquire preliminary data for a successful R01 application more quickly.
Controlling both the types and levels of proteins produced in the cell is of the utmost importance, and factors that regulate the efficiency of protein synthesis are tightly regulated themselves. Defects in the regulation of these factors leads to cancer, heart disease, and other disorders. Information from this project will tell us more about how defects in protein synthesis lead to human diseases, potentially leading to new strategies for early identification and treatment.
| Munoz, Antonio M; Yourik, Paul; Rajagopal, Vaishnavi et al. (2017) Active yeast ribosome preparation using monolithic anion exchange chromatography. RNA Biol 14:188-196 |
