Control of a Plant Translational Regulatory Protein Through Nutrient Signaling

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Gallie The 5'-leader (called omega) of tobacco mosaic viral RNA functions as a translational enhancer. Sequence analysis of a 102 kDa protein, previously identified as a specific omega RNA-binding protein, revealed homology to the HSP101 family and its expression in yeast complemented a thermotolerance defect caused by a deletion of the HSP104 gene. Up to a 20-fold increase in the translation of omega-luc but not luc mRNA was observed in yeast expressing the tobacco HSP101 whereas omega failed to enhance translation in the absence of HSP101. Therefore, HSP101 and omega comprise a two-component translational regulatory mechanism that can be recapitulated in yeast. Analysis of HSP101 function in yeast translation mutants suggested that the initiation factors eIF3 and eIF4G were required for the HSP101-mediated enhancement. The RNA-binding and translational regulatory activities of HSP101 were inactive in respiring cells, following amino acid starvation, or during late-exponential/stationary growth, whereas its thermotolerance function remained unaffected. This has led to a model in which the function of HSP101 as a translational regulator requires activation of its RNA-binding activity which in turn is controlled by nutrient signaling. The first goal of this project is to investigate how HSP101 functions as an RNA-binding protein by identifying the RNA-binding site through deletion and mutation analysis. The second goal will determine how its RNA-binding activity is regulated by nutrient availability. This research seeks to understand how a protein that is induced in response to heat shock (HSP101) regulates the synthesis of tobacco mosaic virus proteins. Since the two components that are responsible for this regulation, an RNA sequence called omega and the tobacco HSP101 protein, are sufficient to recapitulate this regulation when expressed in yeast, this project will examine the role of HSP101 in binding to RNA and regulating translation in yeast. The project will further examine the regulation of HSP101 function in yeast by nutrient availability. Thus, this research will increase our understanding of a mechanism that confers on plants the ability to regulate protein production.