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.
Highlights
The term “folates” refers to a family of enzyme cofactors that carry and chemically activate single carbons at three different oxidation states and function in a metabolic network known as folate-mediated one-carbon metabolism [1]
The Cytoplasmic serine hydroxymethyltransferase (cSHMT) 5Ј-UTR (UTR or a full-length 332-nucleotide 5Ј-UTR (AluUTR)) was placed at the 3Ј-end of the Rluc ORF and 5Ј of the firefly luciferase (FFluc) ORF, which was followed by a 30-nucleotide poly(A) tail (Table 1, template 1)
Translation of the FFluc ORF could potentially occur through three independent mechanisms: 1) mRNA degradation resulting in the formation of a monocistronic FFluc template; 2) failure of the ribosome to dissociate from the template at one of the three tandem termination codons located 3Ј of the Rluc ORF, otherwise known as translation re-initiation; or 3) cap-independent recruitment of the ribosome by the cSHMT 5Ј-UTR serving as an internal ribosome entry site (IRES)
Summary
GCG TCC; the underlined sequence is a BamHI site to aid in cloning into the vector. The reverse primer was 5Ј TA CGGCCG TTA. The cSHMT 3Ј-UTR (635 nucleotides) was amplified (sequence 3Ј of the stop codon to the consensus AAUAAA poly(A) site) from reverse transcribed total RNA isolated from MCF-7 cells (IScript kit, Bio-Rad) by PCR using the following primers: 5ЈTACCCGGGAGGAGCGGGCCCACTCTG-3Ј and 5Ј-TACCCGGGCTGGTTGATTCTCACACC-3Ј. Translation reaction mixtures (25 l) containing 12.5 l of rabbit reticulocyte lysate, amino acid mixture (1 mM each), 1 mM MgOAc, 2 mM dithiothreitol, 100 ng of yeast tRNA, and 35 mM KCl (Flexi rabbit reticulocyte lysate system kit, Promega) were incubated at 30 °C for 15 min with the bicistronic mRNA templates. Cells were either lysed and subjected to SDS-PAGE/immunoblotting analysis to determine knockdown efficiency or used in mRNA transfection experiments as described above
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