Sequence and structure determinants of Drosophila Hsp70 mRNA translation: 5'UTR secondary structure specifically inhibits heat shock protein mRNA translation.

Preferential translation of Drosophila heat shock protein 70 (Hsp70) mRNA requires only the 5'-untranslated region (5'-UTR). The sequence of this region suggests that it has relatively little secondary structure, which may facilitate efficient protein synthesis initiation. To determine whether minimal 5'-UTR secondary structure is required for preferential translation during heat shock, the effect of introducing stem-loops into the Hsp70 mRNA 5'-UTR was measured. Stem-loops of -11 kcal/mol abolished translation during heat shock, but did not reduce translation in non-heat shocked cells. A -22 kcal/mol stem-loop was required to comparably inhibit translation during growth at normal temperatures. To investigate whether specific sequence elements are also required for efficient preferential translation, deletion and mutation analyses were conducted in a truncated Hsp70 5'-UTR containing only the cap-proximal and AUG-proximal segments. Linker-scanner mutations in the cap-proximal segment (+1 to +37) did not impair translation. Re-ordering the segments reduced mRNA translational efficiency by 50%. Deleting the AUG-proximal segment severely inhibited translation. A 5-extension of the full-length leader specifically impaired heat shock translation. These results indicate that heat shock reduces the capacity to unwind 5-UTR secondary structure, allowing only mRNAs with minimal 5'-UTR secondary structure to be efficiently translated. A function for specific sequences is also suggested.     

Translational regulation of Hsp90 mRNA. AUG-proximal 5'-untranslated region elements essential for preferential heat shock translation

Heat shock in Drosophila results in repression of most normal (non-heat shock) mRNA translation and the preferential translation of the heat shock mRNAs. The sequence elements that confer preferential translation have been localized to the 5'-untranslated region (5'-UTR) for Hsp22 and Hsp70 mRNAs (in Drosophila). Hsp90 mRNA is unique among the heat shock mRNAs in having extensive secondary structure in its 5'-UTR and being abundantly represented in the non-heat shocked cell. In this study, we show that Hsp90 mRNA translation is inefficient at normal growth temperature, and substantially activated by heat shock. Its preferential translation is not based on an IRES-mediated translation pathway, because overexpression of eIF4E-BP inhibits its translation (and the translation of Hsp70 mRNA). The ability of Hsp90 mRNA to be preferentially translated is conferred by its 5'-UTR, but, in contrast to Hsp22 and -70, is primarily influenced by nucleotides close to the AUG initiation codon. We present a model to account for Hsp90 mRNA translation, incorporating results indicating that heat shock inhibits eIF4F activity, and that Hsp90 mRNA translation is sensitive to eIF4F inactivation.     

Rapamycin conditionally inhibits Hsp90 but not Hsp70 mRNA translation in <Emphasis Type="Italic">Drosophila</Emphasis>: implications for the mechanisms of Hsp mRNA translation

Rapamycin inhibits the activity of the target of rapamycin (TOR)-dependent signaling pathway, which has been characterized as one dedicated to translational regulation through modulating cap-dependent translation, involving eIF4E binding protein (eIF4E-BP) or 4E-BP. Results show that rapamycin strongly inhibits global translation in Drosophila cells. However, Hsp70 mRNA translation is virtually unaffected by rapamycin treatment, whereas Hsp90 mRNA translation is strongly inhibited, at normal growth temperature. Intriguingly, during heat shock Hsp90 mRNA becomes significantly less sensitive to rapamycin-mediated inhibition, suggesting the pathway for Hsp90 mRNA translation is altered during heat shock. Reporter mRNAs containing the Hsp90 or Hsp70 mRNAs’ 5′ untranslated region recapitulate these rapamycin-dependent translational characteristics, indicating this region regulates rapamycin-dependent translational sensitivity as well as heat shock preferential translation. Surprisingly, rapamycin-mediated inhibition of Hsp90 mRNA translation at normal growth temperature is not caused by 4E-BP-mediated inhibition of cap-dependent translation. Indeed, no evidence for rapamycin-mediated impaired eIF4E function is observed. These results support the proposal that preferential translation of different Hsp mRNA utilizes distinct translation mechanisms, even within a single species.     

Continuous heat shock enhances translational initiation directed by internal ribosomal entry site

Many cellular mRNAs contain internal ribosomal entry sites (IRES) that become functional under conditions of cellular stress, when the rate of protein synthesis for most cellular mRNA is reduced. Internal ribosomal entry increases in response to hypoxia, cell differentiation, apoptosis, gamma irradiation, and heat shock. Heat shock is the principal cellular stress in which general cap-dependent translation is inhibited. On the other hand, heat shock induces the preferential translation of a small class of mRNA, called heat shock protein (HSP) mRNAs, which probably occurs because little or no eIF4F activity is required for their translation. In this study, we found that continuous heat stress enhances expression of the heat shock protein BiP at the level of translation. Interestingly, heat stress also enhanced the viral IRES-dependent translation of encephalomyocarditis virus and hepatitis C virus but not poliovirus. Although several BiP inducers increased BiP protein expression, BiP IRES-dependent translation was enhanced only during heat shock, suggesting that heat shock is a specific inducer for BiP IRES-dependent translation. Taken together, these results indicate that the mechanism of IRES-dependent translation can be used during heat shock and suggest that this translational mechanism may be critical to the survival and proliferation of cells under stress.     

Translational regulation of the synthesis of a major heat shock protein in HeLa cells

The synthesis of a major heat shock protein (HSP 70) was measured in HeLa cells incubated at 42.5 degrees C and then transferred to 37 degrees C or 30 degrees C. After 90 min, synthesis of HSP 70 decreased by 54 and 85%, respectively, whereas HSP 70 mRNA was reduced at most by 20%. Therefore, the reduced synthesis of HSP 70 could not be accounted for by mRNA turnover. HSP 70 was associated with large polyribosomes (6-10 ribosomes) in cells kept at 42.5 degrees C, but with medium or small polyribosomes in cells transferred to 37 degrees C or 30 degrees C (5-6 or 2-3 ribosomes, respectively). Addition of puromycin to these cells resulted in the release of all ribosomes from HSP 70 mRNA, indicating that they were translationally active. The regulation of HSP 70 synthesis was investigated in cell-free systems prepared from heat-shocked or control cells and incubated at 30 degrees C and 42 degrees C. After 5 min at 42 degrees C, the cell-free system from heat-shocked cells synthesized protein at 3 times the rate of the control cell-free system. This difference was in large part due to synthesis of HSP 70. Addition of HSP mRNA to the control cell-free system stimulated protein synthesis at 42 degrees C, but not at 30 degrees C. These findings suggest that translation of HSP 70 mRNA is specifically promoted at high temperature and repressed during recovery from heat shock by regulatory mechanisms active at the level of initiation.