Molecular mechanisms of eukaryotic translational control

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SUMMARY: The level of each protein synthesized in a cell results from both the amount of mRNA available, and the number of ribosomes that initiate translation of that mRNA. Eukaryotic translation initiation factors (eIFs) can reprogram which mRNAs are available and translated to produce different levels of the encoded proteins in response to discrete cellular signals. In fungal species, translational control plays important roles in host evasion and other pathogenesis mechanisms, and in mammalian species, altered levels and modifications of eIFs influence cancer progression and other disease states. A number of protein factors stimulate translation, including the eIF4 factors (4A, B, E, G, and H) that engage the 5’ cap of mRNA and the small subunit of the ribosome, to promote events prior to start site recognition. Ample data support roles of the RNA helicase 4A and cofactors 4B and 4G in unwinding 5’ UTRs to promote translation of structured mRNAs. However, the mRNAs that depend on 4B activity for translation are distinct from those that rely heavily on 4G, even though both factors promote 4A activity. Moreover, in striking contrast to protumorigenic 4A and 4G, increased levels of 4B improved survival of patients with aggressive cancers, and preliminary data directly implicated 4B in opposing metastasis of murine breast cancers. Understanding how 4B mediates translation of specific transcripts could be important for understanding and predicting metastatic phenotypes. Finally, posttranslational modifications of the eIF4 factors are known to modulate translation in mammals, and deregulation is common in disease states. However, it is unclear how modifications of the eIF4 factors regulate translation in fungal species and how this affects expression of specific mRNA pools. This proposal takes on these challenges, investing years of experience characterizing the mechanics of yeast translation initiation and a powerful arsenal of in vitro, genetic, and genome- wide tools. In order to better understand how mRNAs are selected by ribosomes for translation, the objective of this work is to understand how 4B achieves translation versus repression of specific mRNA pools in yeast. Three aims will be pursued: Aim 1. Determine how 4F and 4B interactions affect translation of 4B hyper and hypo-dependent mRNAs. Aim 2. Elucidate yeast 4B- RNA binding preferences. Aim 3. Understand how posttranslational modifications (PTMs) of 4F and 4B impact 4B-specific mRNA control. This proposal will use eIF4B as a model to determine how core translation initiation factors modulate translation versus repression of specific mRNA pools and will shed light on how robust activity of 4B opposes the metastatic phenotype.