Faculty of Engineering and Natural Sciences
HNRNP PROTEINS REGULATE CAT-1 IRES ACTIVITY DURING AMINO ACID STARVATION
Case Western Reserve University
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 transcription, stability and translation of the cat-1 mRNA. Increased translation of the cat-1 mRNA is achieved via an internal ribosome entry site (IRES) within the 5’ leader. 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. It was previously demonstrated by our lab that translation of the uORF unfolds an inhibitory structure in the mRNA leader, eliciting a conformational change that yields an inducible IRES. This study suggested that the structure of the IRES is dynamic and regulation of this RNA structure is a mechanism of translational control. Furthermore, it is shown that the translation efficiency of the endogenous cat-1 mRNA increases by amino acid starvation as evident from the polysome profiles. The lag time between the activation of the 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 as the result of 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. hnRNP L and PTB can modulate cat-1 IRES activity in vivo by means of siRNA based knock-down of expression. 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.
April 26, 2006, 13.40 FENS L056