Figure 4. eEF1A1 binds the 3′UTR of HSP70 mRNA and stabilizes it.

(A) eEF1A1 binds chromatin-associated HSP70 mRNA. Native ChIP samples were IPed using anti-eEF1A1 antibodies and reverse transcribed followed by HSP70 mRNA quantification. Data are represented as mean ± SEM from three independent experiments. (B) eEF1A1 interacts with the HSP70 mRNA in vivo as detected by RNA-IP. The panel shows the RT-PCR products of HSP70 and GAPDH mRNA from control (C) and heat-shocked (HS) cells after IP with indicated antibodies. IgG indicates the mock control. Input is total RNA. (C) Interaction between the HSP70 mRNA 3′UTR and eEF1A1 as detected by RNA-EMSA. 4 μg of purified eEF1A1 were incubated with 105 cpm of the 3′UTR or 5′UTR of HSP70 mRNA or a 200 nt fragment of the β-actin ORF radiolabeled by in vitro transcription. Only the 3′UTR of HSP70 mRNA was shifted by eEF1A1 (red line) and further super-shifted by 4 μg of antibody against eEF1A1 (red star). (D) Knock down of eEF1A1 diminishes HSP70 mRNA stability. Actinomycin D (Ac) was added 30 min after the onset of heat shock. The level of HSP70 and GAPDH mRNA at this time was taken as 100%. Data are represented as mean ± SEM from three experiments. *p < 0.05. (E) eEF1A1-mediates luciferase expression cloned in a HSP70 backbone plasmid. Mock transfected (si:NT) or eEF1A1-knocked down (si:EF) MDA-MB231 cells were transfected with plasmids expressing the SV40- or HSP70-driven luciferase gene fused to the HSP70 3′UTR, and a SV40-renilla luciferase plasmid used as a control. Luciferase activity was measured in cells heat-shocked for 1 hr at 43°C followed by 4 hr of recovery at 37°C. The values were normalized to those of renilla in the same cellular extracts. Bars represent luciferase activity in eEF1A1-deficient cells relative to that obtained for mock-transfected cells. Data are represented as mean ± SEM from three experiments. *p < 0.05.

DOI: http://dx.doi.org/10.7554/eLife.03164.017

Figure 4—figure supplement 1. eEF1A1 binds the 3′UTR of HSP70 mRNA and stabilizes it.

(A) Knock down of eEF1A1 diminishes HSP70 mRNA stability. Actinomycin D (Ac) was added 30 min after the onset of heat shock. The level of HSP70 and GAPDH mRNA at this time point was taken as 100%. Data are represented as mean ± SEM from two experiments. (B) eEF1A1 binds to stem-loop 2 of the HSP70 3′UTR as determined by EMSA. Indicated concentrations of purified eEF1A1 were incubated with 20 kcpm of in vitro transcribed, end labeled HSPA1A 3′UTR RNA (∼0.1 pmol/µl) for 30 min at room temperature. Probe Wt refers to HSPA1A 3′UTR sequence, ΔSL1 is the deletion of nt 7–34, ΔSL2 is the deletion of nt 83–93, 151–158, ΔSL3 is the deletion of nt 217–226, 246–255. M-fold predicted structure for HSPA1A 3′UTR is shown. The stem-loops are marked SL1, SL2, and SL3. The deletion regions are indicated by red rectangles. (C) Determination of the dissociation constant for eEF1A1-HSP70 3′UTR. Experiment was performed as in B.

DOI: http://dx.doi.org/10.7554/eLife.03164.018