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Nutritional Modulation of Gene Expression and Homocysteine Utilization by Vitamin B12

2003; Elsevier BV; Volume: 278; Issue: 23 Linguagem: Inglês

10.1074/jbc.m300845200

1083-351X

Sebastian Oltean, Ruma Banerjee,

Iron Metabolism and Disorders

Vitamins B12, B6, and folic acid converge at the homocysteine metabolic junction where they support the activities of two key enzymes involved in intracellular homocysteine management, methionine synthase (MS) and cystathionine β-synthase. The molecular mechanism for the regulation of homocysteine metabolism by B12 supplementation has been investigated in this study. B12 supplementation does not alter mRNA or protein turnover rates but induces translational up-regulation of MS by shifting the mRNA from the ribonucleoprotein to the polysome pool. The B12-responsive element has been localized by deletion analysis using a reporter gene assay to a 70-bp region located at the 3′ end of the 5′-untranslated region of the MS mRNA. The cellular consequence of the B12 response is a 2- and 3.5-fold increase in the flux of homocysteine through the MS-dependent transmethylation pathway in HepG2 and 293 cells, respectively. It is speculated that B12-induced up-regulation of MS may have evolved as an adaptive strategy for rapidly sequestering an essential and rare nutrient whose availability may have been limited in the evolutionary history of mammals, a problem that is exacerbated by the absence of this vitamin from the plant kingdom. Vitamins B12, B6, and folic acid converge at the homocysteine metabolic junction where they support the activities of two key enzymes involved in intracellular homocysteine management, methionine synthase (MS) and cystathionine β-synthase. The molecular mechanism for the regulation of homocysteine metabolism by B12 supplementation has been investigated in this study. B12 supplementation does not alter mRNA or protein turnover rates but induces translational up-regulation of MS by shifting the mRNA from the ribonucleoprotein to the polysome pool. The B12-responsive element has been localized by deletion analysis using a reporter gene assay to a 70-bp region located at the 3′ end of the 5′-untranslated region of the MS mRNA. The cellular consequence of the B12 response is a 2- and 3.5-fold increase in the flux of homocysteine through the MS-dependent transmethylation pathway in HepG2 and 293 cells, respectively. It is speculated that B12-induced up-regulation of MS may have evolved as an adaptive strategy for rapidly sequestering an essential and rare nutrient whose availability may have been limited in the evolutionary history of mammals, a problem that is exacerbated by the absence of this vitamin from the plant kingdom. Vitamins B12, folic acid, and B6 converge at a single metabolic junction in mammals that is involved in homocysteine detoxification. Elevated levels of homocysteine are a risk factor for cardiovascular diseases (1Refsum H. Ueland P.M. Nygard O. Vollset S.E. Annu. Rev. Med. 1998; 49: 31-62Crossref PubMed Scopus (1819) Google Scholar), neural tube defects (2Mills J.L. McPartlin J.M. Kirke P.N. Lee Y.J. Conle M.R. Weir D.G. Lancet. 1995; 345: 149-151Abstract PubMed Scopus (500) Google Scholar), and Alzheimer's disease (3Clarke R. Smith A.D. Jobst K.A. Refsum H. Sutton L. Ueland P.M. Arch. Neurol. 1998; 55: 1449-1455Crossref PubMed Scopus (1249) Google Scholar). It is estimated that up to 40% of the general population at risk for heart diseases may have elevated levels of homocysteine. Despite these significant statistics, our understanding of homocysteine regulation and how nutrients (viz. vitamins) can modulate flux of homocysteine through competing pathways is poor. Of the three vitamins important for homocysteine metabolism, B12 has the most limited distribution in nature; it is biosynthesized by some bacteria and is absent from the plant kingdom. In humans, it supports the activities of two known enzymes, the cytoplasmic methionine synthase (MS) 1The abbreviations used are: MS, methionine synthase; UTR, untranslated region; CAT, chloramphenicol acetyltransferase; H4folate, tetrahydrofolate. 1The abbreviations used are: MS, methionine synthase; UTR, untranslated region; CAT, chloramphenicol acetyltransferase; H4folate, tetrahydrofolate. and the mitochondrial methylmalonyl-CoA mutase (4Kolhouse J.F. Allen R.H. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 921-925Crossref PubMed Scopus (71) Google Scholar, 5Mellman I.S. Youngdahl-Turner P. Huntington F.W. Rosenberg L.E. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 916-920Crossref PubMed Scopus (53) Google Scholar). MS is a housekeeping enzyme that plays an essential function, and knockout of this gene in mice results in embryonic lethality in the homozygous state (6Swanson D.A. Liu M.L. Baker P.J. Garrett L. Stitzel M. Wu J. Harris M. Banerjee R. Shane B. Brody L.C. Mol. Cell. Biol. 2001; 21: 1058-1065Crossref PubMed Scopus (134) Google Scholar). The ability of vitamin B12 added to the cell culture medium to activate MS was first reported more than three decades ago (7Mangum J.H. North J.A. Biochem. Biophys. Res. Commun. 1968; 32: 105-110Crossref PubMed Scopus (20) Google Scholar, 8Mangum J.H. Murray B.K. North J.A. Biochemistry. 1969; 8: 3496-3499Crossref PubMed Scopus (43) Google Scholar). It is important to emphasize that in these studies cells were exposed to B12 supplementation after having been cultured in "normal" rather than B12-depleted media. Thus, the induction of MS activity by B12 was elicited by vitamin supplementation rather than a transition from B12-deplete to B12-replete medium. Although the molecular basis for this induction was not described, it was suggested that it resulted from conversion of apoenzyme to holoenzyme in the presence of exogenous B12 (9Kamely D. Littlefield J.W. Erbe R.W. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 2585-2589Crossref PubMed Scopus (77) Google Scholar). More recently, we have published evidence that ruled out apoenzyme to holoenzyme conversion as a plausible mechanism for B12-induced activation of MS and demonstrated that although the steady-state levels of mRNA were unaffected, the MS protein levels increased 2–3-fold (10Gulati S. Brody L.C. Banerjee R. Biochem. Biophys. Res. Commun. 1999; 259: 436-442Crossref PubMed Scopus (30) Google Scholar). These results ruled out transcriptional regulation and suggested that the B12 effect may be exerted at a translational level. In principle, the effect of B12 at a posttranscriptional level could occur by enhanced mRNA or protein stability or by increased translational efficiency. This study was designed to distinguish between these possibilities and to determine whether modulation of MS translation efficiency by B12 is mediated by an element in the 5′-UTR of the mRNA. The physiological relevance of B12-dependent gene regulation was demonstrated by the observation that B12 supplementation resulted in enhanced flux of homocysteine through the MS-dependent transmethylation pathway in cultured cells. Materials—Eagle's minimum essential medium, Dulbecco's modified Eagle's medium, hydroxocobalamin, puromycin, actinomycin D, aprotinin, leupeptin, pepstatin, and anti-rabbit IgG (alkaline phosphatase conjugate) were purchased from Sigma. Fetal bovine serum was from HyClone. 293 cells (transformed human epithelial kidney cells) and HepG2 cells (human hepatocellular carcinoma) were from American Type Culture Collection. COS-1 (monkey kidney fibroblast) cells were obtained from Dr. Charles Wood at the University of Nebraska, Lincoln, NE. Restriction enzymes were from Invitrogen or New England Biolabs. [14CH3]H4folate (barium salt, 57 mCi/mmol) was from Amersham Biosciences. Cell Culture Conditions—Cells were grown in Eagle's minimum essential medium supplemented with 10% fetal bovine serum and maintained at 37 °C, 5% CO2. B12 derived from fetal bovine serum is present at an estimated final concentration of ∼125 pm in this medium (9Kamely D. Littlefield J.W. Erbe R.W. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 2585-2589Crossref PubMed Scopus (77) Google Scholar). For B12 induction studies, the cells were grown to 60–80% confluency, and fresh Eagle's minimum essential medium supplemented with 5 mg/liter hydroxocobalamin (or 3.6 μm final concentration) was added. For Western and Northern analyses of MS and for the luciferase reporter studies, cells were harvested from 100-mm plates. [14CH3]H4folate incorporation was examined in cells grown in 60-mm plates. Protein and mRNA turnover experiments were performed in the presence of puromycin and actinomycin, respectively (at final concentrations of 10 μg/ml each), added to the cell culture medium. Western Blot Analysis of MS—Western blot analysis was performed as described previously (10Gulati S. Brody L.C. Banerjee R. Biochem. Biophys. Res. Commun. 1999; 259: 436-442Crossref PubMed Scopus (30) Google Scholar). The bands were quantitated by densitometry using NIH image software. To ensure equal loading, the membranes were also probed with an antibodies against Propionibacterium shermanii methylmalonyl-CoA mutase. Northern Blot Analysis of MS mRNA—Total RNA was isolated using the Trizol reagent (Invitrogen) according to the manufacturer's protocol, and Northern blots were performed as described previously (10Gulati S. Brody L.C. Banerjee R. Biochem. Biophys. Res. Commun. 1999; 259: 436-442Crossref PubMed Scopus (30) Google Scholar). The membrane was then exposed to Biomax film (Eastman Kodak Co.) at–70 °C and developed, and the bands were quantified by densitometry using NIH image software. The band intensities were normalized versus 18 S ribosomal RNA in the same samples. Polysome Analysis—Polysome analysis was performed essentially as described (11Savant-Bhonsale S. Cleveland D.W. Genes Dev. 1992; 6: 1927-1939Crossref PubMed Scopus (148) Google Scholar). Briefly, four 100-mm dishes of COS-1 cells at subconfluent density were used for each polysome distribution analysis. Cells were washed 3 times with ice-cold phosphate-buffered saline containing 100 μg/ml cycloheximide, removed using a cell scraper, and lysed in polysome lysis buffer (100 mm KCl, 5 mm MgCl2, 10 mm HEPES (pH 7.4), 100 μg/ml cycloheximide, 0.5% Nonidet P-40, 20 units/ml RNasin (Promega). Nuclei were pelleted by centrifugation at 12,000 × g at 4 °C for 5 min. Equivalent amounts of RNA were loaded onto each 15–50% (w/v) linear sucrose gradient. Gradients were centrifuged at 39,000 rpm for 2.5 h at 4 °C in a Beckman SW41 rotor, and fractions were collected with an ISCO model 640 density gradient fractionator (ISCO, Inc. Lincoln, NE). Total RNA from each fraction was extracted with phenol/chloroform and reverse transcription-PCR was performed using the SuperScriptTM kit (Invitrogen). cDNA was obtained using gene-specific primers. For MS, a fragment of 340 bases from the 5′-UTR was amplified. For methylmalonyl-CoA mutase, a fragment encompassing the coding sequence was amplified. Products of the reverse transcription-PCR reactions were separated on agarose gels and visualized with ethidium bromide staining. Control experiments were conducted to ensure that the reverse transcription-PCR reactions were in the linear range of amplification by examining the dependence of the product intensity on the number of amplification cycles. Based on this analysis, 25 and 30 cycles of amplification were employed with the MS and methylmalonyl-CoA mutase primers, respectively. The bands were quantified by densitometry using Quantity One (Bio-Rad) software. Three independent experiments were performed. Luciferase and CAT Reporter Constructs—A clone containing the 5′-UTR of MS was generously provided by Dr. Barry Shane (University of California, Berkeley). To follow the effects of the 5′-UTR of MS on a downstream reporter gene, the full-length 5′-UTR of MS or the second half (bases 173–394) were cloned upstream of the luciferase- or CAT-coding regions in the pGL3 or pCAT3-Promoter vectors (Promega), respectively. Pairs of primers for amplification of the full-length 5′-UTR of MS and for the first and second halves of the 5′-UTR of MS were designed as follows: full-length 5′-UTR sense primer, 5′-CCCAAGCTTAAAGGTTCTAAAT-3′, and antisense primer, 5′-CATGCCATGGTTGTCGAGTCTC-3′; first half of 5′-UTR antisense primer, 5′-CATGCCATGGGACCCCGAAAGCCCGCAGCG-3′, and second half of 5′-UTR sense primer, 5′-CCCAAGCTTCCAGCAGTTGCCGCGCCCAG-3′. HindIII and NcoI restriction sites were engineered into the 5′ and the 3′ primers, respectively. After PCR amplification, the DNA fragments were directly cloned into the HindIII/NcoI sites of the pGL3 promoter vector (Promega) so that the MS 5′-UTR was followed by the luciferase start codon. The only difference was a cytosine inserted between the MS 5′-UTR and the luciferase start codon that resulted from the creation of the NcoI site. The pGL3 promoter vector contains an SV40 promoter close to the HindIII site so that 20 bases are added upstream of the MS 5′-UTR in the resulting reporter gene transcript. All PCR reactions were performed using the high fidelity Pfu polymerase (Stratagene) to minimize the risk of introducing unwanted mutations. The sequences of all the constructs were verified by nucleotide sequence determination at the Center for Biotechnology Core Facility (University of Nebraska, Lincoln, NE). All the clones had a single base difference (a G instead of A at position 161) as compared with the reported MS 5′-UTR in Gen-BankTM (accession number U73338). Deletion constructs containing 340, 270, 140, and 70 bases of the 5′-UTR of MS were generated by PCR and cloned into the pGL3-Promoter vector using the HindIII/NcoI sites. For PCR amplification of these fragments, the antisense primer described above was used. The sense primers were designed as follows: fragment 54–394, 5′-CCCAAGCTTTGCCGGTTTTCTCTTGGGTC-3′; fragment 124–394, 5′-CCCAAGCTTCTAGGCCGACACCAAGGACT-3′; fragment 173–394, 5′-CCCAAGCTTAGCCAACGGCAGGCGTCAAA-3′; fragment 254–394, 5′-CCCAAGCTTTTGGCCGTCGTCACCTGTGG-3′. To determine the effects of the 5′-UTR on a second reporter, the full-length 5′-UTR of MS (bases 1–394) and the second half (bases 173–394) were subcloned into the pCAT3-Promoter vector (Promega) using the same cloning strategy described above for the luciferase reporter construct. Transient Transfection and Reporter Assays—Transfection was performed using the LipofectAMINE reagent (Invitrogen) for 293 cells or the GeneJammer (Stratagene) for HepG2 and COS-1 cells. Briefly, 15–20 μg of plasmid DNA was mixed with 80–100 μl of transfection reagent according to the manufacturer's specifications, and the mixture was added to 100-mm plates. 24 h after transfection, B12 was added to half of the plates. At the end of the incubation, two-thirds of each plate were scraped, and cells were centrifuged and saved for total RNA isolation. The remaining one-third of the cells were lysed using the protocol supplied with the luciferase assay system (Promega). The luciferase activity was measured using a luminometer (average of three readings). CAT activity was measured according to the vendor's protocol (Promega). Both luciferase and CAT activities were normalized to total protein concentration in each sample. To account for differences in transfection efficiency (and therefore transcription) between plates, the luciferase and CAT activities were additionally normalized to luciferase and CAT mRNA levels, respectively, as determined by Northern blot analysis. Northern Blot Analysis of Luciferase and CAT mRNA—Total RNA was isolated using the RNAqueaous kit (Ambion) according to the manufacturer's protocol, and Northern blots were performed as described previously (10Gulati S. Brody L.C. Banerjee R. Biochem. Biophys. Res. Commun. 1999; 259: 436-442Crossref PubMed Scopus (30) Google Scholar). 32P-Labeled probes consisting of a DNA fragment encompassing the entire luciferase or CAT open reading frame were purified and labeled with random primers using the Rediprime kit (Amersham Biosciences). Bands were quantified by densitometry using Quantity One (Bio-Rad) software. The band intensities were normalized to the 18 S ribosomal RNA band in each sample to correct for variations in loading quantities. Measurement of [14CH3]-H4folate Incorporation into Proteins—Cells were grown to 60–80% confluency on 60-mm plates, and B12 (5 mg/liter) was added to half of the plates for 24 h. After B12 induction, incorporation of [14CH3]-H4folate into proteins was measured as previously described (12Danishpajooh I.O. Gudi T. Chen Y. Kharitonov V.G. Sharma V.S. Boss G.R. J. Biol. Chem. 2001; 276: 27296-27303Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Cells were incubated for 30 min in methionine-deficient media (Dulbecco's modified Eagle's medium lacking methionine and supplemented with 100 μm homocysteine and 9 μm folic acid). 5 μCi of [14CH3]-H4folate (Amersham Biosciences, 57 mCi/mmol; final medium concentration, 53 μm) was added, and incubation was continued for an additional 2 h. Cells were then washed three times with phosphate-buffered saline and suspended in 1 ml of phosphate-buffered saline. 100 μl from each sample was set aside for protein concentration determination. The remaining suspension was centrifuged, and proteins were precipitated with 500 μl of 5% trichloroacetic acid, incubated on ice for 20 min, heated at 80 °C for 30 min, and then cooled on ice for 30 min. The protein precipitate was washed 3 times with 5% trichloroacetic acid and then suspended in 200 μl of 3 m NaOH, and the amount of radioactivity was measured by liquid scintillation counting. The radioactive content was normalized to total protein concentration in each sample. Statistical Analysis—Each experiment was repeated at least three times. Statistical analysis was performed using one-way analysis of variance (Microcal Origin software), and results were considered significant if the p value was 80%) in the holoenzyme form in 293 cells grown in the presence of normal Eagle's minimum essential medium (10Gulati S. Brody L.C. Banerjee R. Biochem. Biophys. Res. Commun. 1999; 259: 436-442Crossref PubMed Scopus (30) Google Scholar). In addition, we have reported that the relative proportion of the holoenzyme form is unchanged under conditions of B12 supplementation, although the enzyme activity is increased 2.5-fold under these conditions (10Gulati S. Brody L.C. Banerjee R. Biochem. Biophys. Res. Commun. 1999; 259: 436-442Crossref PubMed Scopus (30) Google Scholar). Effect of B12on MS Polysomes—We analyzed the influence of B12 on translation of MS by examining the polysomal association of mRNAs in the presence and absence of B12. As shown in Fig. 3, B12 supplementation elicits a 2.5–3-fold increase in the amplitude of MS polysomes and a corresponding decrease in the proportion of MS mRNA associated with the ribonucleoprotein fraction. These results are consistent with activation of MS translation by B12 supplementation. The magnitude of the increase in MS polysomes is consistent with the previously reported 3.8-fold increase in MS activity induced by B12 in COS-1 cells (10Gulati S. Brody L.C. Banerjee R. Biochem. Biophys. Res. Commun. 1999; 259: 436-442Crossref PubMed Scopus (30) Google Scholar). As a control, the distribution of methylmalonyl-CoA mutase, whose activity is not affected by B12 supplementation, was determined under the same conditions. The amplitude of methylmalonyl-CoA mutase message in the polysomes and ribonucleoprotein pools was unchanged, revealing a specific effect of B12 supplementation on MS mRNA. Changes in the polysome density (i.e. number of ribosomes per mRNA) or amplitude (i.e. number of actively translated mRNAs) are diagnostic of the status of translation from a given message. Activation of protein synthesis can occur via increased recruitment of ribosomes to the mRNA being translated. Alternatively, activation can result due to an increase in the number of mRNA molecules undergoing translation due to a shift in the equilibrium between the inactive (translation suppressed, ribonucleoprotein) and active (translation active, polysome) pools (15Mathews M.B. Sonenberg N. Hershey J.W.B. Mathews M.B. Sonenberg N. Hershey J.W.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 1-31Google Scholar). Role of the 5′-UTR in Mediating B12-dependent Increase in Translational Efficiency—The potential role of B12 in increasing the translational efficiency of MS mRNA was probed with two reporter gene assays. The 5′-UTR of the MS mRNA was chosen for these initial studies because it is unusually long (394 bases) and displays a high potential for formation of secondary structures, suggestive of a role in regulation of translation. Reporter constructs were initially generated with either the full-length leader sequence or with the second half (173–394 bases) of the 5′-UTR inserted upstream of the luciferase or CAT reporter gene (Fig. 4). The respective vectors lacking the 5′ MS-UTR served as one set of controls. Both the full-length 5′-UTR of MS and the second half increased translation of the heterologous reporter gene as assessed by the ratio of the activity of the reporter to its own mRNA levels. The presence of the full-length 5′-UTR of MS between the SV40 promoter and the luciferase open reading frame increased the translational efficiency of luciferase in 293 cells ∼7-fold, whereas the presence of the second half of the 5′-UTR increased translational efficiency ∼2-fold as compared with the pGL3-Promoter vector alone (not shown). The increase in reporter activity upon insertion of the 5′ MS-UTR could be due to the presence of other regulatory elements in this region, including two upstream open reading frames and the possible existence of an internal ribosome entry sequence element affording cap-independent translation initiation. 2S. Oltean and R. Banerjee, unpublished results. The roles of these elements in regulating MS translation and their interactions with B12 are under investigation in our laboratory. To determine whether the B12 effect could be observed in the reporter assays and to exclude a cell-specific response, luciferase activity was determined after transient transfection in two cells lines, HepG2 and 293, 24 h after exposure to B12 (or an equivalent volume of medium in controls). To account for variations in transfection efficiency, the luciferase activities were normalized to the luciferase mRNA levels determined by Northern blot analysis, and equal loading of RNA was confirmed by quantitation of the 18 S rRNA in these samples (Fig. 4). In both 293 (Fig. 4A) and HepG2 (not shown) cells, the presence of B12 induced an increase in luciferase activity with the full-length and second half of the leader sequence. Similarly, in COS-1 cells, a 1.2- and 2.9-fold enhancement of CAT reporter activity was observed with B12 when the full length and the second half of the 5′-UTR respectively, were present (Fig. 4B). In all three cell lines the increase in reporter activity induced by B12 was lower than the increase in MS activity (2.5-, 3.8-, and 13.7-fold increase in 293, COS-1, and HepG2 cells, respectively) reported previously (10Gulati S. Brody L.C. Banerjee R. Biochem. Biophys. Res. Commun. 1999; 259: 436-442Crossref PubMed Scopus (30) Google Scholar). The difference may be attributed to the use of transient transfections in which the full extent of induction is missed or to the boundaries of the B12-responsive element extending beyond those of the 5′ leader used in the present constructs or to long distance interactions with elements that are missing in the reporter constructs. These will be explored in future investigations. Localization of B12-responsive Element—A set of nested deletions extending from the 3′ end of the 5′-UTR were constructed, and their ability to confer responsiveness to B12 in the luciferase reporter construct was tested in COS-1 cells (Fig. 5). With each deletion construct, the luciferase activity was compared in the presence and absence of B12 supplementation to the medium. These experiments localized the B12-responsive element to a 70-bp region extending from the 3′ end of the 5′-UTR. It is interesting to note that deletion of the upstream sequences in the 5′-UTR resulted in a small increase in the magnitude of the B12 effect, as seen in the constructs starting from bp 254 and 324. B12Supplementation Enhances Flux Through the Transmethylation Pathway—The effect of B12-induced increase in MS levels on the flux of homocysteine through the transmethylation pathway was examined by measuring incorporation of [14CH3]-H4folate into proteins. CH3-H4folate is a substrate for only one known enzyme, B12-dependent MS, which transfers the methyl group to homocysteine to give methionine (Fig. 6A). B12 supplementation resulted in a 2–3.5-fold increase in incorporation of radiolabel into proteins in HepG2 and 293 cell lines (Fig. 6B). Three other B12 forms, cyano-, deoxyadenosyl- and methylcobalamin, were equally efficacious in eliciting enhanced flux through the MS-dependent transmethylation pathway (not shown). These results support a physiological relevance for the role of B12 supplementation in increasing flux through the transmethylation pathway and, therefore, in intracellular homocysteine clearance. Translational regulation affords a relatively rapid response to external stimuli, bypassing the steps of transcription, splicing, and translocation. This mode of regulation can lead to both global and selective control of gene expression. Well studied examples of global control include changes that occur in eggs immediately after fertilization and in reticulocytes in response to heme levels. Specific translational control may be achieved by one of a variety of mechanisms including (i) autoregulation via binding of the encoded protein to its mRNA as in thymidylate synthase and dihydrofolate reductase (16Chu E. Koeller D.M. Casey J.L. Drake J.C. Chabner B.A. EWlwood P.C. Zinn S. Allegra C.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8977-8981Crossref PubMed Scopus (325) Google Scholar, 17Chu E. Takimoto C.H. Voeller D. Grem J.L. Allegra C.J. Biochemistry. 1993; 32: 4756-4760Crossref PubMed Scopus (119) Google Scholar, 18Chu E. Voeller D. Koeller D.M. Drake J.C. Takimoto C.H. Maley G.F. Maley F. Allegra C.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 517-521Crossref PubMed Scopus (156) Google Scholar), (ii) by trans-regulatory factors such as the iron-responsive element-binding protein involved in ferritin expression (19Leibold E.A. Munro H.N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2171-2175Crossref PubMed Scopus (552) Google Scholar) or a redox-sensitive protein that binds to a translational enhancer in the 3′-UTR of manganese superoxide dismutase (20Fazzone H. Wangner A. Clerch L.B. J. Clin. Invest. 1993; 92: 1278-1281Crossref PubMed Scopus (36) Google Scholar), or (iii) by a short upstream open reading frame in the 5′-UTR as in ornithine and S-adenosylmethionine decarboxylases (21Shantz L.M. Pegg A.E. Int. J. Biochem. Cell Biol. 1999; 31: 107-122Crossref PubMed Scopus (100) Google Scholar). The response of human MS mRNA to the presence of a nutritional cofactor, B12, appears to be a novel example of specific translational control, since the other B12 enzyme found in humans, methylmalonyl-CoA mutase, is unaffected (10Gulati S. Brody L.C. Banerjee R. Biochem. Biophys. Res. Commun. 1999; 259: 436-442Crossref PubMed Scopus (30) Google Scholar). The results reported here rule out a role for B12 in influencing the rate of MS mRNA or protein turnover (Figs. 1 and 2), and together with our earlier results, which excluded conversion of preexisting apoenzyme to holoenzyme as a plausible mechanism (10Gulati S. Brody L.C. Banerjee R. Biochem. Biophys. Res. Commun. 1999; 259: 436-442Crossref PubMed Scopus (30) Google Scholar), implicate translational regulation. This model is further supported by a B12-induced increase in the amplitude of MS mRNA polysomes with a corresponding decrease in the ribonucleoprotein fraction, which suggests alleviation of translational inhibition or "unmasking" of existing MS mRNAs by the cofactor. The human MS mRNA is >7 kb long with a 3.8-kb open reading frame that is hemmed in by a 394-base-long 5′-UTR and an ∼3-kb-long 3′-UTR (13Li Y.N. Gulati S. Baker P.J. Brody L.C. Banerjee R. Kruger W.D. Hum. Mol. Genet. 1996; 5: 1851-1858Crossref PubMed Scopus (127) Google Scholar, 22Chen L.H. Liu M.-L. Hwang H.-Y. Chen L.-S. Korenberg J. Shane B. J. Biol. Chem. 1997; 272: 3628-3634Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In general, long 5′-UTRs exceeding the average length of 100–140 nucleotides and exhibiting the potential for stable secondary structure formation are deemed to be important in translational regulation. These considerations guided our decision to initially focus on interrogating the role, if any, of the 5′-UTR in modulating the response of human MS mRNA to B12. The extent of translational modulation by the cofactor is modest and varies between 2- and 3-fold in most cell lines that we have tested (10Gulati S. Brody L.C. Banerjee R. Biochem. Biophys. Res. Commun. 1999; 259: 436-442Crossref PubMed Scopus (30) Google Scholar) and leads to an up-regulation rather than repression, which is more commonly observed in examples of translational control of gene expression. Luciferase and CAT reporter constructs implicate the importance of sequences in the 3′ end of the 5′-UTR in modulating the B12 response (Fig. 4). This stretch of the human MS mRNA is predicted to form extensive secondary structures (Fig. 7). The ∼2–3-fold enhancement of MS translational efficiency by B12, albeit modest, is not unexpected for MS, which is a housekeeping enzyme. In another folate cycle enzyme, serine hydroxymethyltransferase, ferritin was reported to increase translation efficiency 1.14–2.16-fold in reporter constructs containing the 5′-UTR (24Oppenheim E.W. Adelman C. Liu X. Stover P.J. J. Biol. Chem. 2001; 276: 19855-19861Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The magnitude of changes for most genes involved in energy metabolism and biosynthesis appears to be ∼2-fold, as revealed by a global analysis of gene profiles associated with aging (23Lee C.-K. Klopp R.G. Weindruch R. Prolla T.A. Science. 1999; 285: 1390-1393Crossref PubMed Scopus (1269) Google Scholar). Recent reports reveal inhibition of translation of genomic RNA in the hepatitis C virus by vitamin B12 (25Lott W.B. Takyar S.S. Tuppen J. Crawford D.H. Harrison M. Sloots T.P. Gowans E.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4916-4921Crossref PubMed Scopus (33) Google Scholar). Toe-printing studies reveal that the vitamin stalls initiation by trapping the 80 S ribosomal complexes on the internal ribosome entry site element (26Takyar S.S. Gowans E.J. Lott W.B. J. Mol. Biol. 2002; 319: 1-8Crossref PubMed Scopus (18) Google Scholar). Translational regulation by B12 has also been documented in prokaryotes, where it represses the btuB gene involved in B12 transport in Escherichia coli and Salmonella typhimurium and the cob biosynthesis operon in S. typhimurium (for review, see Ref. 27Roth J.R. Lawrence J.G. Bobik T.A. Annu. Rev. Microbiol. 1996; 50: 137-181Crossref PubMed Scopus (413) Google Scholar). In contrast to the situation observed with the human gene, the bacterial response is specific for adenosylcobalamin and represents classic feedback regulation for both the transport and synthesis pathways when exogenous B12 levels are high. The proposed model involves long range interactions between a translational enhancer in the 5′-UTR and the region preceding the Shine-Dalgarno sequence, which promotes initiation in the absence of B12 (28Ravnum S. Andersson D.I. Mol. Microbiol. 2001; 39: 1585-1594Crossref PubMed Scopus (49) Google Scholar). In the presence of B12, the conserved "B12 box" interacts with the translational enhancer, relieving the long distance interactions and promoting secondary structure formation in the region encompassing the Shine-Dalgarno sequence, thereby inhibiting initiation. Adenosylcobalamin has been shown to specifically inhibit ribosome binding to the btuB RNA (29Nou X. Kadner R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7190-7195Crossref PubMed Scopus (135) Google Scholar). A similar mode of regulation has been proposed for other bacterial vitamin biosynthesis genes including thiamin (30Miranda-Rios J. Navarro M. Soberon M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9736-9741Crossref PubMed Scopus (191) Google Scholar) and riboflavin (31Gelfand M.S. Mironov A.A. Jomantas J. Kozlov Y.I. Perumov D.A. Trends Genet. 1999; 15: 439-442Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Regulatory proteins that bind to adenosylcobalamin have not been identified in prokaryotes despite intensive efforts, raising the possibility that elements in the mRNAs are the direct sensors of this nutrient. Although this issue has not yet been explored in the eukaryotic system reported in this study, it is interesting to note that RNA aptamers selected for very high and specific binding of cyanocobalamin are known (32Lorsch J.R. Szostak J.W. Biochemistry. 1994; 33: 973-982Crossref PubMed Scopus (175) Google Scholar). The structure of one of these aptamers has revealed the basis of the specificity that underlies the high affinity (Kd = 90 nm) and represents one of the tightest aptamer-small molecule interactions that has been characterized (32Lorsch J.R. Szostak J.W. Biochemistry. 1994; 33: 973-982Crossref PubMed Scopus (175) Google Scholar). Although the existence of B12-induced translational repression in prokaryotes can be rationalized from the standpoint of metabolic economy, the advantage of translational up-regulation in mammals is less obvious. One possibility is that the availability of B12 has been limited through much of our evolutionary history, a problem exacerbated by the absence of this vitamin from the plant kingdom. MS is an essential gene, as evidenced by the embryonic lethal phenotype of MS null mice (6Swanson D.A. Liu M.L. Baker P.J. Garrett L. Stitzel M. Wu J. Harris M. Banerjee R. Shane B. Brody L.C. Mol. Cell. Biol. 2001; 21: 1058-1065Crossref PubMed Scopus (134) Google Scholar), and is absolutely dependent on B12 for its activity. The B12-independent MS found in some bacteria and in plants is absent in mammals. It is striking that the known B12-binding proteins and B12-dependent enzymes found in mammals bind their cofactor avidly, in contrast to many prokaryotic enzymes such as glutamate mutase (33Chen H.P. Marsh E.N. Biochemistry. 1997; 36: 7884-7889Crossref PubMed Scopus (49) Google Scholar). Indeed, one of the tightest protein-ligand interactions that have been measured is for chicken haptocorrin, which displays a binding constant for B12 of 1016m–1 (34Marchaj A. Jacobsen D.W. Savon S.R. Brown K.L. J. Am. Chem. Soc. 1995; 117: 11640-11646Crossref Scopus (26) Google Scholar). It is possible then that translational up-regulation of MS by B12 represents an evolutionary adaptation for rapid and efficient sequestration of a rare nutrient, since increased steady-state levels of MS would lead to increased intracellular B12 concentrations. The demonstration that B12 added to cells grown in normal medium leads to enhanced flux through the transmethylation pathway suggests a beneficial role for B12 supplementation. The increased incorporation of radioactivity from [14CH3]-H4folate to proteins (Fig. 6B) in B12-supplemented medium suggests the potential value of such an intervention in homocysteine-lowering clinical trials by a molecular mechanism that has previously not been recognized.