Roquin is a major mediator of iron-regulated changes to transferrin receptor-1 mRNA stability

Abstract

Transferrin receptor-1 (TfR1) has essential iron transport and proposed signal transduction functions. Proper TfR1 regulation is a requirement for hematopoiesis, neurological development, and the homeostasis of tissues including the intestine and muscle, while dysregulation is associated with cancers and immunodeficiency. TfR1 mRNA degradation is highly regulated, but the identity of the degradation activity remains uncertain. Here, we show with gene knockouts and siRNA knockdowns that two Roquin paralogs are major mediators of iron-regulated changes to the steady-state TfR1 mRNA level within four different cell types (HAP1, HUVEC, L-M, and MEF). Roquin is demonstrated to destabilize the TfR1 mRNA, and its activity is fully dependent on three hairpin loops within the TfR1 mRNA 3'-UTR that are essential for iron-regulated instability. We further show in L-M cells that TfR1 mRNA degradation does not require ongoing translation, consistent with Roquin-mediated instability. We conclude that Roquin is a major effector of TfR1 mRNA abundance.

Keywords: Biological Sciences; Cell Biology; Molecular Biology.

Graphical abstract

Figure 1

Testing the relevance of ZC3H12A-C and RC3H1-2 to the steady-state level of TfR1 mRNA (A) Western analysis of Regnase-1 within the parental HAP1 (wt) and ZC3H12A KO, as described within the transparent methods. (B) Western analysis of the Roquin isoforms within the wt and RC3H1 and -2 KOs. (C) Impact of the CRISPR KOs of the indicated Regnase and Roquin family members on the steady-state TfR1 mRNA level. (D) The ZC3H12A KO increased the abundance of the PTGS2 mRNA, an established Regnase-1 substrate. (E) The iron regulation of TfR1 mRNA abundance is eliminated by the Roquin KO/KD in HAP1 cells, as indicated by the ratio of the TfR1 mRNA abundance under iron deplete (DFO) and rich (FAC) conditions being close to one. Values are relative to the TfR1 mRNA in cells that had not been treated with either the DFO or FAC. (F) The two Roquin isoforms can account for the majority of iron-regulated changes to TfR1 mRNA within the tested cell types, as indicated by the decreased DFO/FAC ratio in the presence of the KDs; nc = negative control siRNA. (G) Western analysis of the Roquin KD in the different cell types. KD efficiencies were calculated from 3 to 5 biological replicates and are indicated below the representative blot. The expression of Roquin is too low in the HUVEC cells to unambiguously quantify the KD by the Western analysis. Cells where indicated were treated with FAC or DFO for 5 hr prior to harvesting. Data are the mean ± standard error of the mean (n = 3–5 biological replicates); ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S1.

Figure 2

The KO of Roquin, but not Regnase-1, increases TfR1 mRNA stability (A) The 5-ethynyl uridine (EU) labeling strategy used to measure the TfR1 mRNA half-life, as described within the transparent methods. (B) Impact of the ZC3H12A-C KOs and RC3H1-2 KO/KD on the TfR1 mRNA half-life. Cells were treated with either 100 μg/mL FAC or 100 μM DFO following removal of the EU label and a brief rinse with fresh media. The TfR1 mRNA abundance was normalized to a RPL4 reference. The t_½ values calculated from the first order rate constants are summarized in Table 1. (C) Western analysis of Roquin obtained from the wildtype (wt) HAP1 cells grown in the presence or absence of iron during the 5 hr period when the majority of the TfR1 mRNA is degraded. (D) Quantification of the Western analysis in (C). (E) The Ferritin light chain (FTL) IRE and a mutation (ΔC33, ΔC39) that inhibits the IRP interaction. (F) The EMSA indicating the complex formed between the radiolabeled IRE (FTL) and the IRP within extracts prepared from wt and Roquin KO/KD cells that had been untreated or treated for 5 hr with either DFO or FAC. A 20-fold molar excess of unlabeled IRE but not the ΔC mutant effectively competes out the interaction. Representative of three biological replicates. Data are the mean ± SEM (n = 3 biological replicates).

Figure 3

Roquin's properties are consistent with the known requirements for iron-regulated TfR1 mRNA instability (A) Secondary structure of the minimized TfR1 3′-UTR instability sequence indicating mutations to the three hairpin loops (I,III,V) that inhibit degradation (red). Potential complications from changes to IRP binding and protection are avoided through single nucleotide deletions to the IRE loops (green). (B) Mutation of each non-IRE hairpin loop increased firefly luciferase activity relative to the wildtype (wt) reporter when expressed in HAP1 cells. KO/KD of Roquin, but not Regnase-1, negates the instability associated with the three TfR1 mRNA hairpin loops. This can be partially rescued by co-expression of Roquin-1 but not with a Roquin mutation (K239A, R260A) previously demonstrated to inhibit its activity. Western analysis of Roquin expression under each condition is indicated below the bar graph. (C) A strong hairpin was inserted 4 nt upstream of the AUG start codon of the firefly luciferase-TfR1 reporter. (D) The hairpin completely inhibits translation of the firefly luciferase-TfR1 reporter. Values are normalized to that of a Renilla luciferase activity encoded on the same plasmid as the firefly luciferase-TfR1 reporter. (E) Effect of the blockage to translation on the abundance of the firefly luciferase reporter mRNA. Mutation of the three essential hairpin loops (I,III,V) indicates the result expected if iron-regulated instability had been inhibited. The abundance of the firefly luciferase mRNA is relative to that of the Renilla luciferase mRNA. L-M cells were treated with 100 μg/mL FAC for 14 hr prior to the assay. Data are the mean ± SEM (n = 3 biological replicates); ∗p < 0.02; ∗∗p < 0.01; ∗∗∗p < 0.001.