Alternative ferritin mRNA translation via internal initiation

Abstract

Ferritin stores and detoxifies an excess of intracellular iron, and thereby plays an important role in the metabolism of this metal. As unshielded iron promotes oxidative stress, ferritin is crucial in maintaining cellular redox balance and may also modulate cell growth, survival, and apoptosis. The expression of ferritin is controlled by transcriptional and post-transcriptional mechanisms. In light of the well-established transcriptional induction of ferritin by inflammatory signals, we examined how ferritin mRNA translation responds to stress conditions. We first used HT1080 fibrosarcoma cells engineered for coumermycin-inducible expression of PKR, a stress kinase that inhibits protein synthesis in virus-infected cells by phosphorylating eIF2α. In this setting, iron triggered partial ferritin mRNA translation despite a PKR-induced global shutdown in protein synthesis. Moreover, iron-mediated ferritin synthesis was evident in cells infected with an attenuated strain of poliovirus; when eIF4GI was cleaved, eIF2α was phosphorylated, and host protein synthesis was inhibited. Under global inhibition of protein synthesis or specific inhibition of ferritin mRNA translation in cells overexpressing PKR or IRP1, respectively, we demonstrate association of ferritin mRNA with heavy polysomes. Further experiments revealed that the 5' untranslated region (5' UTR) of ferritin mRNA contains a bona fide internal ribosomal entry site (IRES). Our data are consistent with the existence of an alternative, noncanonical mechanism for ferritin mRNA translation, which may primarily operate under stress conditions to protect cells from oxidative stress.

FIGURE 1.

De novo ferritin synthesis despite global translational arrest upon eIF2α phosphorylation. HT1080-GyrB-PKR cells were treated with coumermycin (100 ng/mL) and hemin (100 μM) for 6 h. (A) Cell lysates were analyzed by Western blotting with an eIF2α phosphospecific (top) and a β-actin antibody (bottom). (B) Cytoplasmic extracts were fractionated on sucrose gradients with continuous monitoring of A₂₆₀; arrows indicate the heavy polysomes. (C) The cells were metabolically labeled with [³⁵S]methionine/cysteine for 2 h and 1000 μg of cell extracts were subjected to immunoprecipitation with 30 μg of ferritin antibody. Immunoprecipitated material and the IP supernatants were analyzed by SDS-PAGE on a 12% gel. The ferritin bands and the lanes showing global protein synthesis were visualized by autoradiography. (D) Analysis of H-ferritin mRNA from whole-cell lysates by qPCR.

FIGURE 2.

De novo ferritin synthesis in poliovirus-infected cells. (A) HT1080 cells were infected with the Sabin-like type I strain of poliovirus at a multiplicity of infection (MOI) of 10. At the indicated time intervals post-infection (h), cell lysates were prepared and analyzed by Western blotting with eIF4GI (top) and phospho-eIF2α (bottom) antibodies. (B) HT1080 cells were infected with the poliovirus (MOI of 10) for 15 h, treated with hemin for 6 h, and metabolically labeled with [³⁵S]methionine/cysteine for 2 h. Cell extracts (1000 μg) were subjected to immunoprecipitation with 30 μg of ferritin antibody. Immunoprecipitated material and the IP supernatants were analyzed by SDS-PAGE on a 12% gel. The ferritin bands and the lanes corresponding to host and viral protein synthesis were visualized by autoradiography. (C) Analysis of H-ferritin mRNA from whole-cell lysates by qPCR.

FIGURE 3.

Ferritin mRNA associates with the heavy polysomes following treatment with hemin, under conditions of global translational arrest. Cytoplasmic lysates from HT1080-GyrB-PKR cells were subjected to polysome profiling with continuous monitoring of A₂₆₀. The cells were previously either left untreated (A) or treated with 100 ng/mL coumermycin (B), 100 μM hemin (C), or both coumermycin and hemin (D). H-ferritin and β-actin mRNA were analyzed by quantitative real-time PCR. The percentage of H-ferritin (E) and β-actin (F) mRNA in the fractions lighter or heavier than 80S is shown on the graphs corresponding to each treatment.

FIGURE 4.

Association of IRP1_C437S with the polysome-bound ferritin mRNA. (A) The experimental procedure; cell lysates obtained from H1299 cells grown at high density in the presence or absence of tetracycline were subjected to either Western blotting with ferritin and β-actin antibodies (B), or qPCR with H-ferritin, TfR1, and GAPDH specific primers (C), or ultracentrifuged for 1 h to isolate the heavy polysomes from the monosomes. The polysomal pellet was subjected to immunoprecipitation with a FLAG antibody. Ninety pecent of the immunoprecipitate was used for RNA extraction and reverse transcription PCR with H-ferritin, TfR1, and GAPDH-specific primers (D), and 10% of the immunoprecipitate was subjected to Western blotting with a FLAG antibody to document the presence of FLAG-tagged IRP1_C437S in the immunoprecipitate (E).

FIGURE 5.

Evidence that the 5′ UTR of H-ferritin harbors a functional IRES. (A) Schematic diagram of the bicistronic, positive feedback vector used to test for putative IRESs. The transcription of the bicistronic mRNA depends on a minimal promoter containing four copies of the Gal4 upstream activator sequence (4×UAS). The first cistron encodes for Renilla luciferase and the second for the transactivator Gal4/VP16. The 5′ UTR of H-ferritin (Ftn) and the inverted sequence of H-ferritin 5′ UTR (aFtn) were cloned in the intercistronic sequence (ICS). (B) Renilla luciferase activity following transfection of HT1080 cells with control (ICS), Ftn (5′UTR), or aFtn (5′UTR) constructs. The values from three independent experiments (mean ± SD) were normalized with Firefly luciferase activity from a cotransfected construct.

FIGURE 6.

The 5′ UTR of H-ferritin does not possess transcriptional activity. Firefly luciferase activity following transfection of HT1080 cells with (A) the pGL3Basic, pGL3Basic/Ftn(5′UTR), or pGL3Basic/NF-κB constructs, and (B) the pGL3Basic/NF-κB or pGL3Control constructs. The values from three independent experiments (mean ± SD) were normalized with Renilla luciferase activity from cotransfected pRL-TK.

FIGURE 7.

The 5′ UTR of H-ferritin drives cap-independent translation of Firefly luciferase in a bicistronic construct. (A) Schematic diagram of the classic bicictronic construct. The first cistron encodes for Renilla luciferase, preceded by a cap structure and followed by three in-frame stop codons. The second cistron encodes for Firefly luciferase, followed by a poly(A) tail. An intracistronic sequence (ICS) is placed between the two cistrons (B). In vitro transcribed bicistronic RNAs with an ICS consisting of the 5′ UTR of SHMT1, or H-ferritin in sense (Ftn) or antisense orientation (aFtn) were transfected into HT1080 cells. Luciferase activity was monitored in cell lysates; values from three independent experiments (mean ± SD) are shown.