Fig 2: The eIF3 complex interacts with histone mRNAs in vivo. Immunoprecipitation of the endogenous eIF3 complex and its associated RNAs from HEK293FT cells using antibodies against eIF3b (α-eIF3b). A, analysis of the endogenous immunoprecipitated proteins by SDS-PAGE and western blotting using antibodies against the indicated proteins. In: Input (5% of total); (-): Control without antibodies. The position of the molecular weight markers is indicated. B, analysis of mRNAs interacting with eIF3. The relative mRNA enrichment in the eIF3b IP was measured by qRT-PCR and determined by the ΔΔCt method using LDHA mRNA as a normalizer. Circles represent values obtained in three independent experiments and the bars correspond to the mean. c-JUN mRNA (blue bar) is a previously characterized eIF3 target (19). H1, H2A, H2B, H3, and H4 (orange bars) are histone mRNAs. GAPDH, HPRT, PGK1, ACTB, and LDHA are canonical mRNAs and snRNA U2 is a negative control (white bars). Error bars represent the standard deviation of the three independent experiments. The horizontal line represents the level of LDHA mRNA binding (5% in average, normalized to 1). Asterisks indicate statistically significant differences with the LDHA control mRNA. ∗p < 0.05 and ∗∗p < 0.005 based on Student’s t-test.

Fig 3: Physiological effects of the endogenous removal of the 3RE repressive element.(A,B) Representative western blots of (A) FTL and (B) FTH levels in the edited (∆3RE) and WT HepG2 cells under normal iron conditions. Serial dilutions were used in order to better visualize the significance of the changes in FTL and FTH protein abundance. (C, D) Representative western blots of FTL levels in the edited (∆3RE) and WT HepG2 cells under high- or low-iron conditions. Iron donor treatment with FAC at 50 μg/mL for 24 hr, and Iron chelation treatment with DFO at 50 μM for 48 hr. The asterisk (*) indicates that lysate from ∆3RE cells were diluted two-fold, due to the higher overall levels of FTL in these cells. All FTL blots are representative of three or more replicates. (E, F) Determination of the preferential binding of eIF3 to FTL mRNA via EIF3B immunoprecipitation (IP) followed by RNA extraction and RT-qPCR in both (E) HEK293T and (F) HepG2 cell lines. Control mRNAs used to normalize IPs were PSMB6 and ACTB. Error bars represent the standard deviation of duplicate qPCR measurements from representative IP reactions. (G) Determination of IRP1 binding to FTL mRNA in WT (HEK + IRP) and ∆3RE (∆3RE + IRP) HEK293T cells via FLAG immunoprecipitation (IP) followed by RNA extraction and RT-qPCR. The ACTB mRNA was used to control for nonspecific binding to FLAG-tagged IRP. HEK – IRP reflects cells that were not transiently transfected, but were carried through the IP and following experiments. Error bars represent the standard deviation for triplicate measurements from representative IP reactions.10.7554/eLife.48193.022Figure 3—source data 1.Data anlysis of eIF3B and FLAG-tagged IRP1 immunoprecipitations.

Fig 4: Interaction of eIF3 with TCRA and TCRB mRNA 3’-UTR elements mediates a burst in TCR translation in primary human T cells.(A) Schematic of nanoluciferase reporters stably expressed in primary human T cells. The reporters carry the HBB 5’-UTR and WT, ?PAR or R*PAR 3’-UTRs. WT, intact 3’-UTR from either TCRA or TCRB mRNA; ?PAR, 3’-UTR of TCRA or TCRB with the eIF3 PAR-CLIP site deleted; R*PAR, reversed PAR-CLIP sequence in the 3’-UTR of TCRA or TCRB mRNA. (B) Luciferase activity in anti-CD3/anti-CD28 activated T cells stably expressing nanoluciferase constructs described in A, relative to non-activated controls (ACT/NoACT). (C) Changes in nanoluciferase mRNA levels in B, as determined by qRT-PCR. (D) Schematic of immunoprecipitation of eIF3 using an anti-EIF3B antibody (Lee et al., 2015), followed by qRT-PCR to quantify the amount of nanoluciferase mRNA bound to eIF3. (E) Immunoprecipitation as shown in D showing the amount of nanoluciferase mRNA bound to eIF3 in T cells stably transduced with either TCRA (left) or TCRB (right) WT, ?PAR or R*PAR 3’-UTRs after activation with anti-CD3/anti-CD28 antibodies for 1 hr. The percent mRNA bound to anti-EIF3B beads is calculated relative to total mRNA isolated from the cells. (F) Schematic of nanoluciferase reporters stably expressed in primary human T cells. Nanoluciferase reporters carry the HBB 5’-UTR and WT, ?PAR, R*PAR of the TCRA 3’-UTR, or ?PAR of the TCRA 3’-UTR replaced with either a JUN 5’-UTR hairpin or hepatitis C viral (HCV) internal ribosome entry site (domain IIIabc). (G) Luciferase activity in anti-CD3/anti-CD28 activated T cells stably expressing nanoluciferase constructs described in F, relative to non-activated controls. (H) Changes in nanoluciferase mRNA levels in G, as determined by qRT-PCR. (I) Immunoprecipitation as shown in D to quantify the amount of nanoluciferase mRNA bound to eIF3 in T cells stably expressing nanoluciferase constructs in F after activation with anti-CD3/anti-CD28 antibodies for 0.5, 1, 3, and 5 hr. The percent mRNA bound to anti-EIF3B beads is calculated relative to total mRNA isolated from the cells. All experiments were carried out in triplicate (three separate wells per condition), with mean and standard deviations shown. All the primary human T cell experiments were performed using two donors and data from one representative donor is shown.

Fig 5: eIF3 remains bound to the coding sequences (CDS) of pan-mRNAs independent of their 5’-UTR and 3’-UTR elements in actively translating ribosomes.(A) Schematic outlining the RNase H-based assay of eIF3 interactions with mRNAs in polysomes. DSP refers to the dithiobis (succinimidyl propionate) crosslinking agent. Oligos, DNA oligos designed for RNase H-mediated targeting and cleavage of specific mRNAs. (B) Strategy for detecting mRNA fragments released by RNase H digestion. Red arrows denote DNA oligos for RNase H-mediated targeting of mRNAs. RT-qPCR primers (black) were used to detect the CDS regions of the mRNAs. (C) Amount of eIF3-bound mRNA co-immunoprecipitated by an anti-EIF3B antibody (Lee et al., 2015), from polysome fractions of Jurkat cells treated with RNase H and oligos targeting the CDS-UTR junctions (red arrows diagrammed in panel B). (D) Amount of eIF3-bound mRNA co-immunoprecipitated with IgG beads, from polysome fractions of Jurkat cell lysate treated with RNase H and oligos targeting the CDS-UTR junctions. (E) Amount of eIF3-bound mRNA co-immunoprecipitated by the anti-EIF3B antibody, from polysome fractions of Jurkat cell lysate treated only with RNase H. (F) Amount of eIF3-bound mRNA co-immunoprecipitated by an anti-EIF3B antibody, from polysome fractions of primary human T cells treated with RNase H and oligos targeting the CDS-UTR junctions (red arrows diagrammed in panel B). (G) Amount of eIF3-bound mRNA co-immunoprecipitated with IgG beads, from polysome fractions of primary human T cell lysate treated with RNase H and oligos targeting the CDS-UTR junctions. (H) Amount of eIF3-bound mRNA co-immunoprecipitated by the anti-EIF3B antibody, from polysome fractions of primary human T cell lysate treated only with RNase H. In panels C–H, the percentage is relative to the amount of total mRNA present in the polysome fraction prior to immunoprecipitation. All the immunoprecipitation experiments in panels C–H were carried out in biological duplicate with one technical triplicate shown (n = 3, with mean and standard deviations shown). The primary human T cell experiment was done using two donors.