A novel FADS1 isoform potentiates FADS2-mediated production of eicosanoid precursor fatty acids
2012; Elsevier BV; Volume: 53; Issue: 8 Linguagem: Inglês
10.1194/jlr.m025312
ISSN1539-7262
AutoresWoo Jung Park, Kumar S.D. Kothapalli, Holly T. Reardon, Peter Lawrence, Shu-Bing Qian, J. Thomas Brenna,
Tópico(s)Lipid metabolism and biosynthesis
ResumoThe fatty acid desaturase (FADS) genes code for the rate-limiting enzymes required for the biosynthesis of long-chain polyunsaturated fatty acids (LCPUFA). Here we report discovery and function of a novel FADS1 splice variant. FADS1 alternative transcript 1 (FADS1AT1) enhances desaturation of FADS2, leading to increased production of eicosanoid precursors, the first case of an isoform modulating the enzymatic activity encoded by another gene. Multiple protein isoforms were detected in primate liver, thymus, and brain. In human neuronal cells, their expression patterns are modulated by differentiation and result in alteration of cellular fatty acids. FADS1, but not FADS1AT1, localizes to endoplasmic reticulum and mitochondria. Ribosomal footprinting demonstrates that all three FADS genes are translated at similar levels. The noncatalytic regulation of FADS2 desaturation by FADS1AT1 is a novel, plausible mechanism by which several phylogenetically conserved FADS isoforms may regulate LCPUFA biosynthesis in a manner specific to tissue, organelle, and developmental stage. The fatty acid desaturase (FADS) genes code for the rate-limiting enzymes required for the biosynthesis of long-chain polyunsaturated fatty acids (LCPUFA). Here we report discovery and function of a novel FADS1 splice variant. FADS1 alternative transcript 1 (FADS1AT1) enhances desaturation of FADS2, leading to increased production of eicosanoid precursors, the first case of an isoform modulating the enzymatic activity encoded by another gene. Multiple protein isoforms were detected in primate liver, thymus, and brain. In human neuronal cells, their expression patterns are modulated by differentiation and result in alteration of cellular fatty acids. FADS1, but not FADS1AT1, localizes to endoplasmic reticulum and mitochondria. Ribosomal footprinting demonstrates that all three FADS genes are translated at similar levels. The noncatalytic regulation of FADS2 desaturation by FADS1AT1 is a novel, plausible mechanism by which several phylogenetically conserved FADS isoforms may regulate LCPUFA biosynthesis in a manner specific to tissue, organelle, and developmental stage. arachidonic acid alternative transcript eicosapentaenoic acid cytomegalovirus endoplasmic reticulum fatty acid desaturase FADS1 alternative transcript 1 fatty acid methyl ester long chain polyunsaturated fatty acid open reading frame polyadenylation rapid amplification of cDNA ends transcription start site Alternative transcripts (AT) produced by RNA processing events, such as alternative splicing, alternative transcription initiation, and alternative polyadenylation, generate diverse mRNA isoforms cotranscriptionally from a single gene (1Poulos M.G. Batra R. Charizanis K. Swanson M.S. 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Structure, function, and dietary regulation of delta6, delta5, and delta9 desaturases.Annu. Rev. Nutr. 2004; 24: 345-376Crossref PubMed Scopus (826) Google Scholar). FADS1 (Δ5-desaturase), FADS2 (Δ6-desaturase/Δ8-desaturase), and FADS3 arose evolutionarily by gene duplication events and are clustered within the 100 kb region on human chromosome 11. They share similar exon/intron organization and contain four well-conserved motifs (cytochrome b5 and three histidine boxes) (17Nakamura M.T. Nara T.Y. Structure, function, and dietary regulation of delta6, delta5, and delta9 desaturases.Annu. Rev. Nutr. 2004; 24: 345-376Crossref PubMed Scopus (826) Google Scholar, 18Park W.J. Kothapalli K.S. Lawrence P. Tyburczy C. Brenna J.T. An alternate pathway to long-chain polyunsaturates: the FADS2 gene product Delta8-desaturates 20:2n-6 and 20:3n-3.J. Lipid Res. 2009; 50: 1195-1202Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In the LCPUFA biosynthetic pathway, FADS1 catalyzes endogenous synthesis of arachidonic acid (AA; 20:4n-6) and eicosapentaenoic acid (EPA; 20:5n-3) from dihomo-γ-linolenic acid (DGLA; 20:3n-6) and eicosatetraenoic acid (ETA; 20:4n-3), respectively (Fig. 1). AA and EPA serve as precursors for eicosanoids (19Calder P.C. Polyunsaturated fatty acids and inflammatory processes: new twists in an old tale.Biochimie. 2009; 91: 791-795Crossref PubMed Scopus (562) Google Scholar). Numerous meta-analyses and genome-wide association studies (GWAS) identified genetic variants at FADS1 loci to be associated with human phenotypes influencing glucose homeostasis, cholesterol and triglyceride levels, and resting heart rate (20Dupuis J. Langenberg C. Prokopenko I. Saxena R. Soranzo N. Jackson A.U. Wheeler E. Glazer N.L. Bouatia-Naji N. Gloyn A.L. et al.New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk.Nat. Genet. 2010; 42: 105-116Crossref PubMed Scopus (1650) Google Scholar–22Eijgelsheim M. Newton-Cheh C. Sotoodehnia N. de Bakker P.I. Muller M. Morrison A.C. Smith A.V. Isaacs A. Sanna S. Dorr M. et al.Genome-wide association analysis identifies multiple loci related to resting heart rate.Hum. Mol. Genet. 2010; 19: 3885-3894Crossref PubMed Scopus (103) Google Scholar). Human FADS1 (23Cho H.P. Nakamura M. Clarke S.D. Cloning, expression, and fatty acid regulation of the human delta-5 desaturase.J. Biol. Chem. 1999; 274: 37335-37339Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar) spans 17.2 kb of genomic DNA, shares 61% and 52% identity with FADS2 and FADS3, respectively, and encodes a protein of 444 amino acids with a molecular mass of 52.0 kDa (24Marquardt A. Stohr H. White K. Weber B.H. cDNA cloning, genomic structure, and chromosomal localization of three members of the human fatty acid desaturase family.Genomics. 2000; 66: 175-183Crossref PubMed Scopus (228) Google Scholar). The classical FADS1 transcript has been shown to be highly expressed in the liver, brain, and heart (23Cho H.P. Nakamura M. Clarke S.D. Cloning, expression, and fatty acid regulation of the human delta-5 desaturase.J. Biol. Chem. 1999; 274: 37335-37339Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). Even though FADS1 operates on both n-6 and n-3 PUFA, only a single transcript has been identified to date. However, currently in the National Center for Bioinformatics Information (NCBI) database, human FADS1 (GenBank accession NM_013402) is represented with an open reading frame (ORF) that encodes a 501 amino acid peptide. This new larger peptide contains 65 amino acids more than the classical Δ5-desaturase protein (444 amino acids), yet the function of it is not known. We recently showed the first alternative transcripts for FADS2 and FADS3, which were generated by alternative splicing events and expressed in a tissue-specific manner in a primate (neonate baboon) (25Park W.J. Kothapalli K.S. Reardon H.T. Kim L.Y. Brenna J.T. Novel fatty acid desaturase 3 (FADS3) transcripts generated by alternative splicing.Gene. 2009; 446: 28-34Crossref PubMed Scopus (40) Google Scholar, 26Park W.J. Reardon H.T. Tyburczy C. Kothapalli K.S. Brenna J.T. Alternative splicing generates a novel FADS2 alternative transcript in baboons.Mol. Biol. Rep. 2010; 37: 2403-2406Crossref PubMed Scopus (16) Google Scholar). They are conserved in several mammals and the chicken, and they exhibit reciprocal changes in gene expression in response to human neuronal cell differentiation (25Park W.J. Kothapalli K.S. Reardon H.T. Kim L.Y. Brenna J.T. Novel fatty acid desaturase 3 (FADS3) transcripts generated by alternative splicing.Gene. 2009; 446: 28-34Crossref PubMed Scopus (40) Google Scholar–27Brenna J.T. Kothapalli K.S. Park W.J. Alternative transcripts of fatty acid desaturase (FADS) genes.Prostaglandins Leukot. Essent. Fatty Acids. 2010; 82: 281-285Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). To investigate whether FADS1 is also subject to alternative splicing, we performed both 5′ and 3′ rapid amplification of cDNA ends (RACE) using gene-specific primers from baboon FADS1 (GenBank accession EF531577). Here, we show unambiguous evidence of the existence of several FADS1 mRNA isoforms generated by alternative transcription initiation, alternative selection of poly(A) sites, and internal exon deletions resulting from alternative splicing. Our studies reveal that a FADS1 isoform enhances FADS2 activity, the first known function for a FADS isoform and the first example of an AT from one gene regulating the activity coded by an adjacent gene. These data are consistent with Δ6-desaturase enhancement of desaturase activity by a lipoprotein-like protein reported in the 1980s (28Leikin A.I. Brenner R.R. Regulation of linoleic acid delta 6-desaturation by a cytosolic lipoprotein-like fraction in isolated rat liver microsomes.Biochim. Biophys. Acta. 1986; 876: 300-308Crossref PubMed Scopus (21) Google Scholar). Additionally, we present the detection of protein isoforms in neonate tissues and mammalian cells, protein isoform expression and fatty acid changes during cell differentiation, and isoform-specific subcellular localization, and findings that all three FADS genes are translated at similar levels in mouse embryonic fibroblasts. Studies on baboons were approved by the Cornell University and Texas Biomedical Research Institute (formerly the Southwest Foundation for Biomedical Research) Institutional Animal Care and Use Committees. Neonate baboon liver tissue from a 12-week-old baboon treated with RNAlater and stored at −80°C since necropsy was used to isolate total RNA, and the RNA quality was assessed as described previously (25Park W.J. Kothapalli K.S. Reardon H.T. Kim L.Y. Brenna J.T. Novel fatty acid desaturase 3 (FADS3) transcripts generated by alternative splicing.Gene. 2009; 446: 28-34Crossref PubMed Scopus (40) Google Scholar). First-strand 5′ RACE-ready cDNA and 3′ RACE-ready cDNA was prepared per the manufacturer's recommended protocol provided with SMARTerTM RACE cDNA amplification kit (Clontech Laboratories, Mountain View, CA). We performed both 5′ and 3′ RACE using baboon FADS1 (GenBank accession EF531577) gene-specific primers to identify the transcription initiation, splicing, and poly(A) sites. For the 5′ RACE reactions, we used the antisense primer 5′-TGGAAG TGCATGTGGTTCCACCAA-3′, and for the 3′ RACE reactions, we used the sense primer 5′-TGTGTTCTT CCTGC TGTA CCTG CT-3′. The PCR reactions were performed by using the Advantage 2 PCR enzyme system. 5′ and 3′ RACE-amplified products were run on 2% agarose gels. Several products were seen on the gel. Each amplified product was sliced carefully from the gel, and DNA was extracted using PureLinkTM gel extraction kit (Invitrogen, Carlsbad, CA). The gel-purified products were cloned into pGEM-T Easy vector (Promega) and sequenced at Cornell University Life Sciences Core Laboratories Center. Three human cell lines, SK-N-SH neuroblastoma (NB), MCF7 breast cancer, and HepG2 hepatocellular carcinoma cells, were grown in DMEM/F-12, MEM-α, and MEM/EBSS media with 10% FBS (media and serum obtained from HyClone), respectively, at 37°C in a humidified environment with 5% CO2. At confluence, the cells were harvested for RNA and protein extraction. RNA was isolated using the QIAshredder and RNeasy Mini kit (Qiagen, Valencia, CA). Protein extract was carried out using RIPA lysis buffer (Thermo Scientific, IL). Protein concentrations were determined by a bicinchoninic acid (BCA) assay (Thermo Scientific). NB cell differentiation assays were carried out in serum free DMEM/F-12 with 1× N-2 supplement (Invitrogen) as described earlier (25Park W.J. Kothapalli K.S. Reardon H.T. Kim L.Y. Brenna J.T. Novel fatty acid desaturase 3 (FADS3) transcripts generated by alternative splicing.Gene. 2009; 446: 28-34Crossref PubMed Scopus (40) Google Scholar). The open reading frame of FADS transcripts (FADS1, FADS2, FADS1AT1) were cloned into a pcDNA3.1 expression vector containing cytomegalovirus (CMV) promoter (Invitrogen). FADS1AT1 and empty vector stable MCF7 cells were dosed with 100 μM of albumin-bound 18:2n-6 or 20:3n-6 fatty acid and were incubated for 24 h. For cotransfection studies, MCF7 cells stably expressing FADS1AT1 or empty vector were transfected with equal amounts of FADS1 or FADS2 DNA using Lipofectamine LTX (Invitrogen). Twenty-four hours after cotransfection, the cells were incubated with 100 μM of albumin-bound 18:2n-6 or 20:3n-6 fatty acid for additional 24 h. After incubation, the cells were washed twice with 1× PBS and removed by trypsinization. Then fatty acids were analyzed. Fatty acid methyl ester (FAME) preparation and structural identification of FAME was carried out as described earlier (18Park W.J. Kothapalli K.S. Lawrence P. Tyburczy C. Brenna J.T. An alternate pathway to long-chain polyunsaturates: the FADS2 gene product Delta8-desaturates 20:2n-6 and 20:3n-3.J. Lipid Res. 2009; 50: 1195-1202Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Briefly, cells were isolated and total fatty acids hydrolyzed and converted to FAME by a one-step reaction mixture. Structures were identified by gas chromatography covalent adduct chemical ionization tandem mass spectrometry (GC-CACI-MS/MS), which provides positive structural assignments of double-bond positions for all monoene and homoallylic FAME, for low abundance FAME (29Michaud A.L. Diau G.Y. Abril R. Brenna J.T. Double bond localization in minor homoallylic fatty acid methyl esters using acetonitrile chemical ionization tandem mass spectrometry.Anal. Biochem. 2002; 307: 348-360Crossref PubMed Scopus (55) Google Scholar). Quantitative analysis was performed with GC coupled to a flame ionization detector using an equal weight mixture for response factor calibration. Expression levels of FADS1 transcripts was measured using cDNA from nine normal tissues from a 12-week-old baboon neonate and three human cell lines by RT-PCR. cDNA from baboon tissues is from a previous study (25Park W.J. Kothapalli K.S. Reardon H.T. Kim L.Y. Brenna J.T. Novel fatty acid desaturase 3 (FADS3) transcripts generated by alternative splicing.Gene. 2009; 446: 28-34Crossref PubMed Scopus (40) Google Scholar). RT-PCR analysis was performed using primers designed from unique regions specific for each transcript. (Primer sequences are provided upon request.) RT-PCR reactions were carried out using 1 μM of each primer, 0.25 mM each of dNTPs, 1.5 mM MgCl2, and AmpliTaq II (ABI, Foster City, CA) in a final volume of 30 μl. Thermal cycling conditions were as follows: initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 65°C for 45 s, and extension at 72°C for 1 min, with a final extension at 72°C for 5 min. PCR products were separated on 2% agarose gels. Total protein from baboon neonate liver, brain, and thymus tissue lysates and cultured cells was resolved by SDS-PAGE (Bio-Rad, Hercules, CA). The samples were transferred to nitrocellulose membranes and probed with the FADS1 and β-actin antibodies. FADS1 antibody (cat. #AV42384) was purchased from Sigma-Aldrich (St. Louis, MO). β-actin and the secondary antibodies were from LiCor Biosciences (Lincoln, NE). Immuno blots were imaged and immunofluorescence signal was detected by using LiCor Odyssey infrared imaging system as directed by the manufacturer (LiCor Biosciences). To determine the localization of FADS1 and FADS1AT1, the coding regions were cloned into pEGFP-N1 vector driven by the CMV promoter (pEFADS1 and pEFADS1AT1, respectively). NB cells were transfected with the constructed vectors using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA), and organelle-specific stains MitoTracker Red CMXRos or ER-tracker Blue-White DPX (Molecular Probes, Invitrogen) were used to stain organelles. Live cells were visualized with an inverted Meta Confocal Microscope (LSM 510 META, Zeiss). Mitochondria were isolated from the transfected NB cells using mitochondria isolation kit (Thermo Scientific). Western blot was performed using GFP antibody (Abcam), COX IV (inner mitochondrial membrane-specific) antibody (Cell Signaling), and PDI (endoplasmic reticulum-specific) antibody (Cell Signaling). MEF cells were first treated with cycloheximide (100 µg/ml) for 3 min at 37°C to free the translating ribosomes on mRNAs. Cells were then harvested by ice-cold polysome lysis buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2, 100 µg/ml cycloheximide, 5 mM DTT, 20 U/ml SUPERaseIn, and 2% Triton X-100) followed by profiling using 10–50% sucrose gradient. Polysome fractions were collected and treated with RNase I to digest the mRNA segments not protected by ribosomes. The ribosome-protected fragments were enriched and converted into a DNA library suitable for Illumina sequencing. Qualified sequencing reads were aligned to the cDNA database using SOAP2-based mapping software allowing up to two mismatches. We cloned and sequenced the entire coding region of baboon FADS1 (GenBank accession EF531577). The baboon FADS1 coding sequence shares 97% identity with the human FADS1 sequence as well as exon architecture. The 5′ and 3′ RACE was performed using FADS1 gene-specific primers. 5′ RACE PCR experiments generated four products. The long base pair (bp) product was prominent on the ethidium bromide-stained agarose gel, whereas the remaining three products were weakly visualized. All four gel-purified products were sequenced, and the Basic Local Alignment Search Tool (BLAST) confirmed the products (750 bp, 537 bp, 430 bp, and 403 bp) as FADS1 amplicons. Promoter and TSS prediction was performed using Neural Network Promoter Prediction software (30Reese M.G. Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome.Comput. Chem. 2001; 26: 51-56Crossref PubMed Scopus (691) Google Scholar). For the 750 bp sequenced product, the promoter region ranging from −95 bp to −45 bp from the first translation start site was identified with a minimum promoter score of 0.54; the TSS (TSS1) is located at −55 bp from the first translation start site ("TSS1" for the classical transcript FADS1CS). The 750 bp amplicon closely corresponds to previously reported human sequences (GenBank accessions AK027522 and AF084558). The 537 bp amplicon arose because of internal splicing within the distal end of exon 1, followed by a 81 bp N-terminal or 5′ end sequence extension, resulting in an alternative exon ("E1A"). The promoter region (minimum promoter score of 0.27) for this amplicon ranged from −106 bp to −56 bp, and the TSS (TSS2) is located −66 bp from the first translation start site ("TSS2" for the alternative transcript FADS1AT1). In this amplicon, the initial 81 bases (−136 bp to −55 bp) from the first translational start site are strikingly similar to a newly identified sequence from the NEDO splicing variants project (GenBank accession AK299762), which is also highly similar to FADS2. Internal splicing within exon 2 gives rise to 430 bp and 403 bp amplicons. They are located at +224 and +251 of the baboon FADS1 gene (GenBank accession EF531577) and have very short 5′ UTR. We refer to this cluster as "TSS3." We performed 3′ RACE to detect alternative selection of poly(A) sites and splice variants. Seven amplicons with varying lengths were detected on agarose gels. The sizes of the sequenced amplicons were 1114 bp, 1156 bp, 1460 bp, 1606 bp, 1620 bp, 2603 bp and 3750 bp. BLAST (http://www.ncbi.nlm.nih.gov/BLAST) searches confirmed the products as FADS1 amplicons. Six out of the seven amplicons differ only in the 3′ UTR length because of alternative use of poly(A) sites. The 2603 bp amplicon has several exon deletions resulting from internal exon splicing and closely (99% identity) resembles a Rhesus Macaque sequence (GenBank acc# AC194778). In silico analysis of the 5′/3′ RACE sequences for FADS1 show that they have at least four 5′ UTRs and several 3′ UTRs of varying lengths (GenBank accessions JF518968, JF518969, JF518970, JF518971, JF518972, JF518973, JF518974, and JF518975). Putative coding regions of these transcripts were predicted using ORF finder software. The four conserved functional features of most PUFA desaturases are an N-terminal cytochrome b5 domain and three histidine motifs (HXXXH, HXXHH, and QXXHH) conserved from humans to microalga (31Sperling P. Ternes P. Zank T.K. Heinz E. The evolution of desaturases.Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 73-95Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). The amplicon that starts at TSS1 can uniquely be translated from the exon 1 to produce a protein product with 444 amino acids, retaining all four conserved desaturase domains: a cytochrome b5 domain and three histidine motifs. However, the in-frame translation start site for TSS2 and TSS3 amplicons, located within exon 2, all have identical protein-coding sequences. They produce 360 amino acid proteins lacking the N-terminal cytochrome b5 domain (Fig. 2). We also found a 2,603 bp transcript generated by internal exon deletions using 3′ RACE. This transcript had a truncated exon 6, skipped exons 7–11, and truncated exon 12. For this splice variant, ORF finder predicts a 270 amino acid protein using TSS1. The 270 amino acid protein retains the cytochrome b5 domain and the first two histidine motifs, losing the third due to splicing (Fig. 2). Our screening did not identify the novel NCBI-annotated transcript that codes for a predicted 501 amino acid protein. Stable FADS1AT1 MCF7 cells were generated to determine the functional role of novel FADS1AT1 isoform. When incubated for 24 h with 20:3n-6, a natural FADS1 substrate, cells stably expressing FADS1AT1 showed no catalytic activity toward 20:3n-6 (Fig. 3A, B). When transiently transfected with FADS1, these cells showed enhanced conversion of 20:3n-6 to 20:4n-6 compared with nontransfected cells, but FADS1AT1 had no effect on the activity of FADS1 (Fig. 3C, D). Table 1 shows that 20:3n-6 → 20:4n-6 was 10.35 ± 0.01% in FADS1 + vector cells, compared with 11.05 ± 0.04% in FADS1 + FADS1AT1 cells, yielding a ratio of 1.07-fold, not significantly different from 1.TABLE 1Conversion ratio of cotransfected vector-only and FADS1AT1 cells with FADS1SubstrateReactionProduct/Substrate (%) Mean ± SDRatioFADS1 + VectorFADS1 + FADS1AT1(FADS1 + FADS1AT1) / (FADS1 + Vector)20:3n-6Δ5-desaturase (20:3n-6 → 20:4n-6)10.35 ± 0.0111.05 ± 0.041.07 Open table in a new tab When stable FADS1AT1 MCF7 cells were incubated for 24 h with 18:2n-6, a natural FADS2 substrate, no desaturation activity was observed in either control (vector only), or FADS1AT1 cells (Fig. 4A, B). Control and stable FADS1AT1 cells were then transiently transfected with FADS2 DNA and incubated with 18:2n-6. FADS2-transfected vector-only cells gained Δ6-desaturase activity to generate the expected 18:3n-6 product, and also converted 20:2n-6 to 20:3n-6 via Δ8-desaturation (Fig. 4C). When FADS1AT1 cells were transfected with FADS2 DNA, production of 18:3n-6 via Δ6-desaturation more than doubled (Fig. 4D). Moreover, the Δ8-desaturation product 20:3n-6 synthesized via Δ8-desaturation of 20:2n-6 also increased significantly, while apparently depleting 20:2n-6 substrate. Table 2 shows that 18:2n-6 → 18:3n-6 was 3.1 ± 0.4% in FADS2 + vector cells compared with 7.8 ± 1.9% in FADS2 + FADS1AT1 cells, a ratio of 2.5-fold. We also found that 18:2n-6 is elongated to 20:2n-6 and Δ8-desaturated to 20:3n-6, with an apparent conversion of 45 ± 2% in FADS2 + vector cells compared with 71 ± 0.2% in FADS2 + FADS1AT1 cells, a ratio of 1.6-fold.TABLE 2Conversion ratio of cotransfected vector-only and FADS1AT1 cells with FADS2SubstrateReactionProduct/Substrate (%) Mean ± SDRatioPFADS2 + VectorFADS2 + FADS1AT1(FADS2 + FADS1AT1) / (FADS2 + Vector)18:2n-6Δ6-desaturase (18:2n-6 → 18:3n-6)3.1 ± 0.47.8 ± 1.92.50.08Δ8-desaturase (20:2n-6 → 20:3n-6)45 ± 270.5 ± 0.21.60.004 Open table in a new tab To determine tissue-specific expression levels of the transcripts initiated from TSS1 (FADS1CS) and TSS2 (FADS1AT1), we performed RT-PCR analysis using transcript-specific primers. The transcripts initiated from TSS3 have very short 5′ UTRs embedded within the exon 2; it was not possible to check the expression levels of these transcripts as the sequence overlaps with the TSS1 transcripts. FADS1CS expression was higher than FADS1AT1 in all the tissues and human cells examined (Fig. 5A, B). No amplification product was detected in skeletal muscle, and expression in spleen was very low for FADS1AT1. A trace FADS1AT1 band was visible in MCF7 and SK-N-SH (NB) cells. To determine whether the newly identified FADS1 mRNA transcripts encode a protein expressed in tissue, we performed immunoblot analysis using protein lysates extracted from baboon neonate liver, brain, and thymus tissues and three human transformed cells (HepG2, MCF7, and NB). An immunogen directed against the C-terminal third histidine motif was selected because the new putative protein isoform does not contain the N-terminal cytochrome b5 domain; this immunogen was predicted to recognize both the classical and the putative isoforms. We detected two prominent (42 and 48 kDa) and two faint (54 and 65 kDa) produ
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