The transport of amino acids is essential for cellular metabolism and occurs through specific carrier systems. Tile regulation of amino acid transport activity is required for amino acid homeostasis. This regulation is essential during the physiological stress condition of limited amino acid and glucose availability. The latter causes the misfolding of proteins in the endoplasmic reticulum triggering the unfolded protein response (UPR). Cells respond to limitation of these two nutrients by the simultaneous global decrease in protein synthesis (via phosphorylation of the translation initiation factor elF2alpha) and the selective increase of synthesis of proteins that are critical for cell survival. Cat-1 is the major high affinity transporter of the amino acids arginine and lysine. Amino acid and glucose availability regulate expression of cat-1 at many levels, including gene transcription, stability and the translation of the mRNA. In this proposal the mechanism of regulation of cat-1 mRNA levels by amino acid availability and the UPR will be determined. Our hypothesis is that the temporal expression of transcription factors and RNA binding proteins during stress leads to the transient induction of cat-1 gene expression. We propose that this induction involves three steps. (i) Increased cat-1 gene transcription (ii) escape/compensation of the nonsense mediated mRNA decay (NMD) pathway and (iii) increased stability of the cat-1 mRNA. Coordinate regulation of all these steps leads to the accumulation of sufficient cat-1 mRNA to support translation during conditions of cellular translation attenuation. A novel mechanism is proposed, suggesting that the global decrease in protein synthesis during nutritional stress is the signaling pathway for transcriptional induction of cat-1 gene expression via a network of transcription factors and promoter elements.
Our Specific Aims are: (1 and 2). Characterize cis DNA sequences and transcription factors that regulate cat-1 gene transcription during amino acid availability and the UPR. (3). Determine regulation of the cat-1 gene by the nonsense mediated mRNA decay (NMD) pathway. (4). Determine the mechanism via which the nuclear/cytoplasmic shuttling protein HuR regulates cat-1 mRNA levels during limited amino acid supply and the UPR. (5). Determine the interactions between cat-1 transcription, mRNA stabilization/translation and transport activity in response to nutrient availability using cat-1 (-/-) embryonic fibroblast cells. Because the cat-1 gene is regulated by nutrients at three distinct levels (transcription, mRNA stability and translation), the studies described here will be a prototype for future research on nutritional control of gene expression.
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