A Ferritin-responsive Internal Ribosome Entry Site Regulates Folate Metabolism

Abstract

Cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme levels are elevated by the expression of the heavy chain ferritin (H ferritin) cDNA in cultured cells without corresponding changes in mRNA levels, resulting in enhanced folate-dependent de novo thymidylate biosynthesis and impaired homocysteine remethylation. In this study, the mechanism whereby H ferritin regulates cSHMT expression was determined. cSHMT translation is shown to be regulated by an H ferritin-responsive internal ribosome entry site (IRES) located within the cSHMT mRNA 5'-untranslated region (5'-UTR). The cSHMT 5'-UTR exhibited IRES activity during in vitro translation of bicistronic mRNA templates, and in MCF-7 and HeLa cells transfected with bicistronic mRNAs. IRES activity was depressed in H ferritin-deficient mouse embryonic fibroblasts and elevated in cells expressing the H ferritin cDNA. H ferritin was shown to interact with the mRNA-binding protein CUGBP1, a protein known to interact with the alpha and beta subunits of eukaryotic initiation factor eIF2. Small interference RNA-mediated depletion of CUGBP1 decreased IRES activity from bicistronic templates that included the cSHMT 3'-UTR in the bicistronic construct. The identification of this H ferritin-responsive IRES represents a mechanism that accounts for previous observations that H ferritin regulates folate metabolism.

Hemoglobin concentration is inversely associated with erythrocyte folate concentrations in Colombian school-age children, especially among children with low vitamin B12 status

“…These studies suggest that red cells might enhance the uptake of folate from serum in the presence of iron deficiency. Heavy-chain ferritin increases folate catabolism (Woeller et al, 2007); therefore, low ferritin in iron deficiency might spare folate by decreasing its catabolism (Suh et al, 2001). However, the prevalence of iron deficiency according to plasma ferritin concentration in our study was low, and we did not find any interactions between ferritin and folate concentrations in relation to hemoglobin concentrations.…”

“…Given the cross-sectional nature of the study, the inverse association between folate and hemoglobin concentrations might also represent an effect of iron on folate metabolism (Vitale et al, 1966;Woeller et al, 2007). In the course of iron deficiency, serum folate concentration has been shown to be reduced (Vitale et al, 1966), while erythrocyte folate concentration is increased (Omer et al, 1970;Saraya et al, 1973); in addition, treatment with iron could result in decreased erythrocyte folate concentration (Omer et al, 1970).…”

A UV-responsive Internal Ribosome Entry Site Enhances Serine Hydroxymethyltransferase 1 Expression for DNA Damage Repair

“…A, shown is the bicistronic construct used to quantify SHMT1 IRES activity. It consists of (in the 5Ј to 3Ј direction) a cap analog, the Rluc reporter gene followed by three sequential in-frame stop codons, the human SHMT1 5Ј-UTR, which contains the IRES element, the Fluc reporter gene, the human SHMT1 3Ј-UTR, which was shown to stimulate SHMT1 IRES activity (26), and a 30-nucleotide poly(A) tail. B, untreated (light bars) and UV-treated (dark bars) cells were transiently transfected with the bicistronic construct in A.…”

“…Transfections-The generation of pSP64 poly(A) DNA templates containing the Renilla and Firefly luciferase reporter genes and either the SHMT1 5Ј-UTR, SHMT1 5Ј-UTR, and 3Ј-UTR, the reverse complement of the SHMT1 5Ј-UTR (reverse UTR), or the mouse SHMT1 5Ј-UTR is described elsewhere (26). The DNA templates were linearized with EcoRI and purified using the Roche Applied Science PCR clean-up column.…”

“…We have previously shown that the SHMT1 5Ј-UTR contains an IRES whose activity is stimulated by heavy chain ferritin (H ferritin), heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2) (55), and CUG-binding protein 1 (CUGBP1) (26). However, the responsiveness of the IRES to stress stimuli that inhibit cap-dependent translation has not been explored.…”