Abstract
Adaptation to amino acid deficiency is critical for cell survival. In yeast, this adaptation involves phosphorylation of the translation eukaryotic initiation factor (eIF) 2alpha by the kinase GCN2. This leads to the increased translation of the transcription factor GCN4, which in turn increases transcription of amino acid biosynthetic genes, at a time when expression of most genes decreases. Here it is shown that translation of the arginine/lysine transporter cat-1 mRNA increases during amino acid starvation of mammalian cells. This increase requires both GCN2 phosphorylation of eIF2alpha and the translation of a 48-amino acid upstream open reading frame (uORF) present within the 5'-leader of the transporter mRNA. When this 5'-leader was placed in a bicistronic mRNA expression vector, it functioned as an internal ribosomal entry sequence and its regulated activity was dependent on uORF translation. Amino acid starvation also induced translation of monocistronic mRNAs containing the cat-1 5'-leader, in a manner dependent on eIF2alpha phosphorylation and translation of the 48-amino acid uORF. This is the first example of mammalian regulation of internal ribosomal entry sequence-mediated translation by eIF2alpha phosphorylation during amino acid starvation, suggesting that the mechanism of induced Cat-1 protein synthesis is part of the adaptive response of cells to amino acid limitation.
Highlights
Adaptation to amino acid deficiency is critical for cell survival
This increase requires both GCN2 phosphorylation of eIF2␣ and the translation of a 48-amino acid upstream open reading frame present within the 5-leader of the transporter mRNA. When this 5-leader was placed in a bicistronic mRNA expression vector, it functioned as an internal ribosomal entry sequence and its regulated activity was dependent on uORF translation
Amino Acid Starvation Induces Translation Mediated by the cat-1 5Ј-Leader in Monocistronic mRNAs via a Mechanism That Involves eIF2␣ Phosphorylation—The results described in Figs. 1– 4 demonstrate that the IRES activity of the cat-1 5Ј-leader increases in amino acid-depleted cells in a manner dependent
Summary
Generation of Expression Vectors—All the cat-1 5Ј-UTR-containing bicistronic mRNA expression vectors were generated by PCR amplification of the cat-1 5Ј-UTR cDNAs using the SalI/NcoI site of the pSVCAT/ICS/LUC plasmid (12) by replacing the ICS DNA sequence. The monocistronic expression vectors, cat-1 5Ј-UTRf/LUC and cat-1 5Ј-UTRmutf/LUC, were generated by cloning the SalI/XbaI cat-1 5Ј-UTRf/LUC and cat-1 5Ј-UTRmutf/LUC fragments from the bicistronic vectors into the EcoRI/XbaI site of the pUHD10 –3 vector. In this vector, transcription is directed by the minimal cytomegalovirus. All vectors contained the corresponding inserts within the XbaI/HindIII site of pCDNA3 In this vector, transcription is directed by the cytomegalovirus promoter and the selectable marker gene was replaced with the CD2 cell surface marker gene (14). Details of the phosphorylation status of the eIF2␣s and regulation of translation have been described (18, 19)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
