Mechanisms of Post-transcriptional Regulation of Cat-1 Gene Expression by Amino Acid Starvation
The cationic amino acid transporter, cat-1, is a high affinity transporter of the essential amino acids arginine and lysine. Induced expression of the cat-1 gene is essential for cell survival during nutritional stress. Amino acid starvation induces coordinate increases in the stability and translation of the cat-1 mRNA. It is shown here that increased mRNA stability is due to an 11 nucleotide AU-rich element (ARE) within the distal 217 bases of the 3’-untranslated region (UTR) of the 7.9 kb cat-1 mRNA, which is able to confer mRNA stabilization when it is present in the chimeric mRNA. This element binds to the nucleo-cytoplasmic protein HuR whose cytoplasmic concentration increases during amino acid starvation. In addition to mRNA stabilization, the adaptive response to nutritional stress also involves increased translation of cat-1 mRNA via an internal ribosome entry site (IRES). Induction of cat-1 IRES activity requires both translation of a small upstream open reading frame (uORF) within the IRES and phosphorylation of the translation initiation factor eIF2?. We show here that translation of the uORF unfolds an inhibitory structure in the mRNA leader, eliciting a conformational change that yields an inducible IRES. This study suggests that the structure of the IRES is dynamic and regulation of this RNA structure is a mechanism of translational control. The lag time between the activation of cat-1 IRES and the length of amino acid starvation led us to propose that the remodeled leader by uORF translation is stabilized by putative trans-acting factors that are either synthesized or modified by eIF2? phosphorylation. hnRNP L and PTB are among the proteins identified as potential trans-acting factors. The binding specificity of these hnRNP proteins were confirmed by in vitro binding assays. Furthermore, it is shown that increased cytoplasmic localization of hnRNP L and PTB proteins during amino acid starvation paralleled increased binding to cat-1 leader. It is shown here that hnRNP L can modulate cat-1 IRES activity in vivo by means of over-expression or siRNA based knock-down of hnRNP L expression. We show here that the translation efficiency of the endogenous cat-1 mRNA increased by amino acid starvation. Altogether our data further support the role of hnRNP L and PTB in IRES-mediated translation and these proteins are involved in cat-1 IRES regulation during nutritional stress.
School:Case Western Reserve University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:au rich element mrna stability translational control ires amino acid starvation itaf
Date of Publication:01/01/2005