Translation of cIAP2 mRNA Is Mediated Exclusively by a Stress-Modulated Ribosome Shunt

ABSTRACT During cellular stress, translation persists or increases for a number of stress-responsive proteins, including cellular inhibitor of apoptosis 2 (cIAP2). The cIAP2 transcript includes a very long (2.78-kb) 5′ untranslated region (UTR) with an unusually high number of upstream AUGs (uAUGs), i.e., 64, and a stable predicted secondary structure (ΔG ≅ −620 kcal/mol) that should completely block conventional scanning-dependent translation initiation. This region did not facilitate internal ribosome entry in vitro or when RNA reporter transcripts were transfected into cells. However, several structural features within the cIAP2 5′ UTR were observed to be nearly identical to those required for ribosome shunting in cauliflower mosaic virus RNA and are well conserved in cIAP2 orthologs. Selective mutation revealed that the cIAP2 mRNA mediates translation exclusively via ribosome shunting that bypasses 62 uAUGs. In addition, shunting efficiency was altered by stress and was greatly facilitated by a conserved RNA folding domain (1,470 to 1,877 nucleotides upstream) in a region not scanned by shunting ribosomes. This arrangement suggests that regulation of cIAP2 shunting may involve recruitment of RNA binding proteins to modulate the efficiency of translation initiation.

Translational Regulation of Angiotensin Type 1a Receptor Expression and Signaling by Upstream AUGs in the 5′ Leader Sequence

Rat angiotensin type 1a receptor (AT1aR) is regulated by four upstream AUGs present in the 5′ leader sequence (5′-LS). Disruption of all four upstream AUGs (QM) results in 2–3-fold higher levels of angiotensin type 1 receptor (AT1R) densities in transiently transfected rat aortic smooth muscle cells (A10 cells) and stably transfected Chinese hamster ovary cells. Cells expressing QM have 5-fold higher levels of angiotensin II-induced inositol phosphate production than wild type (WT). Polysome analysis showed that QM mRNA is present in heavier fractions than the WT transcript, and 5.7-fold more AT1R protein is produced byin vitrotranslation from QM transcripts compared with WT transcripts. The AT1aR comprises 3 exons. Exon 3 (E3) encodes the entire open reading frame and 3′-untranslated region. Exons 1 and 2 (E1 and E2) and 52 nucleotides of E3 encode the 5′-LS. The AUGs in both exons contribute to the inhibitory effect on AT1R expression but not to the same degree. Disruption of the AUGs in exon 2 (DM2) relieves half of the inhibition, whereas disruption of the AUGs in exon 1 (DM1) is without effect. Disruption of the AUGs in exon 2 results in levels of receptor expression and translation that are indistinguishable from the alternative splice variant E1,3, which we previously showed was more efficiently translated than the E1,2,3 transcript. Individual mutations revealed that only the fourth AUG increased AT1R translation. In conclusion, all four AUGs present in the 5′-LS function cumulatively to suppress AT1aR expression and signaling by inhibiting translation. These data also show that both AUGs in E2 contribute to the inhibitoryciselement present in this alternatively spliced exon.