Abstract
Regulation of homocysteine, a sulfur-containing amino acid that is a risk factor for cardiovascular diseases, is poorly understood. Methionine synthase (MS) is a key enzyme that clears intracellular homocysteine, and its activity is induced by its cofactor, vitamin B12, at a translational level. In this study, we demonstrate that translation of MS, which has a long and highly structured 5'-untranslated region, is initiated from an internal ribosome entry site (IRES), which is modulated by B12. The minimal IRES element spans 71 bases immediately upstream of the initiation codon. Electrophoretic mobility shift analysis reveals the presence of a B12 -dependent protein-RNA complex and suggests the possibility that B12-dependent increase of IRES efficiency is mediated via a protein. Modulation of the IRES-dependent translation of an essential gene by the cofactor of the encoded enzyme represents a novel example of a gene-nutrient interaction.
Highlights
The activity of Methionine synthase (MS) in cells cultured in normal medium is enhanced by supplementation with vitamin B12, an observation that was first reported over 30 years ago [12]
Translation of the first but not the second cistron (Fig. 1B, upper panel) is strongly inhibited by the presence of the hairpin in the CAT/MS 5Ј-UTR/LUC construct and in the positive control containing the BIP-internal ribosome entry site (IRES) (Fig. 1B, lower panel). These results provide evidence that translation of the second cistron occurs by initiation in the intercistronic region and are consistent with the presence of an IRES element in the MS 5Ј-UTR
Our studies reveal a novel mechanism of translational regulation of human MS that is IRES-dependent and is modulated by its cofactor, B12
Summary
Materials—Eagle’s MEM (minimum essential medium), OHCbl, AdoCbl, MeCbl, and CNCbl were purchased from Sigma. Cell Culture Conditions—Cells were grown in Eagle’s MEM supplemented with 10% fetal bovine serum and incubated at 37 °C, 5% CO2. B12 derived from fetal bovine serum is present at a concentration of ϳ125 pM in this medium. For B12 induction studies, the cells were grown to 60 – 80% confluency, and fresh medium supplemented with 5 mg literϪ1 OHCbl (3.6 M final concentration) was added. The pSVCAT/ICS/LUC vector contains 400 nucleotides of antisense antennapedia cDNA of Drosophila melanogaster [17] in the intercistronic (ICS) region and was used as a negative control for IRES-mediated translation.
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