Internal ribosome entry site drives cap-independent translation of reaper and heat shock protein 70 mRNAs in Drosophila embryos

Abstract

Translation is a sensitive regulatory step during cellular stress and the apoptosis response. Under such conditions, cap-dependent translation is reduced and internal ribosome entry site (IRES)-dependent translation plays a major role. However, many aspects of how mRNAs are translated under stress remain to be elucidated. Here we report that reaper mRNA, a pro-apoptotic gene from Drosophila melanogaster, is translated in a cap-independent manner. In Drosophila mutant embryos devoid of the eukaryotic initiation factor 4E (eIF4E), reaper transcription is induced and apoptosis proceeds. In vitro translation experiments using wild-type and eIF4E mutant embryonic extracts show that reporter mRNA bearing reaper 5 untranslated region (UTR) is effectively translated in a cap-independent manner. The 5UTR of reaper exhibits a high degree of similarity with that of Drosophila heat shock protein 70 mRNA, and both display IRES activity. Studies of mRNA association to polysomes in embryos indicate that both reaper and heat shock protein 70 mRNAs are recruited to polysomes under apoptosis or thermal stress. Our data suggest that heat shock protein 70 and reaper, two antagonizing factors in apoptosis, use a similar mechanism for protein synthesis.

Different modes of translation for hid, grim and sickle mRNAs in Drosophila

“…We corroborated the IRES activity of hid and grim by inserting their 5 0 UTR sequences into the dicistronic reporter FLuc/cad/RLuc, which also shows low background of translation of the second cistron 14 ( Figure 5a). We used m 7 GpppGcapped dicistronic mRNAs for in vitro translation (Figure 5b).…”

“…As was observed for the capdependent Actin5C mRNA, polysome-associated sickle mRNAs decreased during heat-shock conditions (P/U 2.2 and 1.5, respectively, in untreated conditions to P/U o 1 during heat-shock conditions). On the contrary, as it was observed for rpr and hsp70 mRNAs, whose cap-independent translation is driven by an IRES element 14 and Figure 6d), hid mRNA move from the untranslated fractions in the untreated condition (P/U ratio 1.3) to polysome-associated fractions during heat-shock conditions (P/U 1.9). grim mRNA does not change the P/U ratio, indicating that it is still maintained in the polysome fraction despite the inhibition of translation by heat shock.…”

“…14 We performed double in situ hybridization using specific probes for each gene and a balancer-specific probe to identify embryos that contain a wild-type copy of eIF4E-1,2 from homozygous eIF4E-1,2 mutant embryos. We found that rpr, hid and, to a lesser extent, sickle mRNA were upregulated and widespread expressed in the homozygous eIF4E-1,2 mutant embryos (Herná ndez et al…”

“…Indeed, Drosophila rpr and hsp70 5 0 UTRs have a high content of adenines (45-50%); 14,33 as well as the 5 0 UTR Hsp83 (38%), which suffices to confer efficient translation upon heatshock by decreasing the likelihood of secondary structure formation. 34 Although no blocks of similarity between hid or grim with hsp70 5 0 UTR exist, as it the case of rpr 5 0 UTR, 14 they are all rich in adenines. hid, rpr, grim and sickle 5 0 UTR have 50, 45, 37 and 34% adenine content, respectively, and they are translated in this order of efficiency in heat-shocked extracts.…”

“…14 rpr mRNA is translated in a capindependent manner by an IRES 14 and several RNA-binding proteins have been identified as potential translational regulators. 15 Here we studied the mechanism of translation of the proapoptotic genes grim, hid and sickle.…”