A novel 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) splice variant with an alternative exon 1 potentially encoding an extended N-terminus

BMC Molecular Biology

Armin P Piehler³  Jens P Berg⁴  Runa M Grimholt²  Marianne K Kringen¹  Camilla Stormo² 

[1] Department of Pharmacology, Oslo University Hospital, Ullevål, P.O box 4956, Nydalen, Oslo, 0424, Norway;Department of Medical Biochemistry, University of Oslo and Oslo University Hospital, Ullevål, P.O box 4956, Nydalen, Oslo, 0424, Norway;Fürst Medical Laboratory, Søren Bullsvei 25, Oslo, NO-1051, Norway;Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, P.O box 1171, Blindern, Oslo, 0318, Norway

关键词: Cholesterol;    Statin;    Alternative splicing;    Transcription;    HMG-CoA;    3-hydroxy-3-methylglutaryl-coenzyme A reductase;   

Others  :  1091531
DOI  :  10.1186/1471-2199-13-29

 received in 2012-01-03, accepted in 2012-07-11,  发布年份 2012

【 摘 要 】

Background

The major rate-limiting enzyme for de novo cholesterol synthesis is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). HMGCR is sterically inhibited by statins, the most commonly prescribed drugs for the prevention of cardiovascular events. Alternative splicing of HMGCR has been implicated in the control of cholesterol homeostasis. The aim of this study was to identify novel alternatively spliced variants of HMGCR with potential physiological importance.

Results

Bioinformatic analyses predicted three novel HMGCR transcripts containing an alternative exon 1 (HMGCR-1b, -1c, -1d) compared with the canonical transcript (HMGCR-1a). The open reading frame of the HMGCR-1b transcript potentially encodes 20 additional amino acids at the N-terminus, compared with HMGCR-1a. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to examine the mRNA levels of HMGCR in different tissues; HMGCR-1a was the most highly expressed variant in most tissues, with the exception of the skin, esophagus, and uterine cervix, in which HMGCR-1b was the most highly expressed transcript. Atorvastatin treatment of HepG2 cells resulted in increased HMGCR-1b mRNA levels, but unaltered proximal promoter activity compared to untreated cells. In contrast, HMGCR-1c showed a more restricted transcription pattern, but was also induced by atorvastatin treatment.

Conclusions

The gene encoding HMGCR uses alternative, mutually exclusive exon 1 sequences. This contributes to an increased complexity of HMGCR transcripts. Further studies are needed to investigate whether HMGCR splice variants identified in this study are physiologically functional.

【 授权许可】

   
2012 Stormo et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Maron DJ, Fazio S, Linton MF: Current perspectives on statins. Circulation 2000, 101:207-213.
• [2]Goldstein JL, Brown MS: Regulation of the mevalonate pathway. Nature 1990, 343:425-430.
• [3]Johnson JM, Castle J, Garrett-Engele P, Kan Z, Loerch PM, Armour CD, Santos R, Schadt EE, Stoughton R, Shoemaker DD: Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science 2003, 302:2141-2144.
• [4]DeBose-Boyd RA: Feedback regulation of cholesterol synthesis: sterol-accelerated ubiquitination and degradation of HMG CoA reductase. Cell Res 2008, 18:609-621.
• [5]Horton JD, Goldstein JL, Brown MS: SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 2002, 109:1125-1131.
• [6]Medina MW, Gao F, Naidoo D, Rudel LL, Temel RE, McDaniel AL, Marshall SM, Krauss RM: Coordinately regulated alternative splicing of genes involved in cholesterol biosynthesis and uptake. PLoS One 2011, 6:e19420.
• [7]Burkhardt R, Kenny EE, Lowe JK, Birkeland A, Josowitz R, Noel M, Salit J, Maller JB, Pe’er I, Daly MJ: Common SNPs in HMGCR in micronesians and whites associated with LDL-cholesterol levels affect alternative splicing of exon13. Arterioscler Thromb Vasc Biol 2078, 2008:28.
• [8]Medina MW, Gao F, Ruan W, Rotter JI, Krauss RM: Alternative splicing of 3-hydroxy-3-methylglutaryl coenzyme A reductase is associated with plasma low-density lipoprotein cholesterol response to simvastatin. Circulation 2008, 118:355-362.
• [9]Istvan ES, Deisenhofer J: Structural mechanism for statin inhibition of HMG-CoA reductase. Science 2001, 292:1160-1164.
• [10]Istvan ES, Palnitkar M, Buchanan SK, Deisenhofer J: Crystal structure of the catalytic portion of human HMG-CoA reductase: insights into regulation of activity and catalysis. EMBO J 2000, 19:819-830.
• [11]Medina MW, Krauss RM: The role of HMGCR alternative splicing in statin efficacy. Trends Cardiovasc Med 2009, 19:173-177.
• [12]UCSC Genome Browser http://genome.ucsc.edu/ webcite.
• [13]Rozen S, Skaletsky H: Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 2000, 132:365-386.
• [14]Piehler AP, Grimholt RM, Ovstebo R, Berg JP: Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes. BMC Immunol 2010, 11:21. BioMed Central Full Text
• [15]Vandesompele J, De PK, Pattyn F, Poppe B, Van RN, De PA, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3:RESEARCH0034.
• [16]geNorm http://medgen.ugent.be/~jvdesomp/genorm/ webcite.
• [17]Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001, 29:e45.
• [18]Protein knowledgebase (UniProtKB) http://www.uniprot.org/ webcite.
• [19]Engfelt WH, Masuda KR, Paton VG, Krisans SK: Splice donor site mutations in the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene cause a deficiency of the endoplasmic reticulum 3-hydroxy-3-methylglutaryl coenzyme A reductase protein in UT2 cells. J Lipid Res 1998, 39:2182-2191.
• [20]Liscum L, Finer-Moore J, Stroud RM, Luskey KL, Brown MS, Goldstein JL: Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum. J Biol Chem 1985, 260:522-530.
• [21]Keller GA, Barton MC, Shapiro DJ, Singer SJ: 3-Hydroxy-3-methylglutaryl-coenzyme A reductase is present in peroxisomes in normal rat liver cells. Proc Natl Acad Sci U S A 1985, 82:770-774.
• [22]Keller GA, Pazirandeh M, Krisans S: 3-Hydroxy-3-methylglutaryl coenzyme A reductase localization in rat liver peroxisomes and microsomes of control and cholestyramine-treated animals: quantitative biochemical and immunoelectron microscopical analyses. J Cell Biol 1986, 103:875-886.
• [23]Porcellini E, Calabrese E, Guerini F, Govoni M, Chiappelli M, Tumini E, Morgan K, Chappell S, Kalsheker N, Franceschi M, et al.: The hydroxy-methyl-glutaryl CoA reductase promoter polymorphism is associated with Alzheimer’s risk and cognitive deterioration. Neurosci Lett 2007, 416:66-70.
• [24]Kovacs WJ, Faust PL, Keller GA, Krisans SK: Purification of brain peroxisomes and localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Eur J Biochem 2001, 268:4850-4859.
• [25]Breitling R, Krisans SK: A second gene for peroxisomal HMG-CoA reductase? A genomic reassessment. J Lipid Res 2002, 43:2031-2036.
• [26]Davuluri RV, Suzuki Y, Sugano S, Plass C, Huang TH: The functional consequences of alternative promoter use in mammalian genomes. Trends Genet 2008, 24:167-177.
• [27]Wang H, Li R, Hu Y: The alternative noncoding exons 1 of aromatase (Cyp19) gene modulate gene expression in a posttranscriptional manner. Endocrinology 2009, 150:3301-3307.
• [28]Melamud E, Moult J: Stochastic noise in splicing machinery. Nucleic Acids Res 2009, 37:4873-4886.
• [29]Feingold KR, Wiley MH, Moser AH, Lau DT, Lear SR, Siperstein MD: De novo sterologenesis in intact primates. J Lab Clin Med 1982, 100:405-410.
• [30]Lampe MA, Burlingame AL, Whitney J, Williams ML, Brown BE, Roitman E, Elias PM: Human stratum corneum lipids: characterization and regional variations. J Lipid Res 1983, 24:120-130.
• [31]Schiefelbein D, Goren I, Fisslthaler B, Schmidt H, Geisslinger G, Pfeilschifter J, Frank S: Biphasic regulation of HMG-CoA reductase expression and activity during wound healing and its functional role in the control of keratinocyte angiogenic and proliferative responses. J Biol Chem 2008, 283:15479-15490.
• [32]Ayoubi TA, Van De Ven WJ: Regulation of gene expression by alternative promoters. FASEB J 1996, 10:453-460.
• [33]Keller L, Murphy C, Wang HX, Fratiglioni L, Olin M, Gafvels M, Bjorkhem I, Graff C, Meaney S: A functional polymorphism in the HMGCR promoter affects transcriptional activity but not the risk for Alzheimer disease in Swedish populations. Brain Res 2010, 1344:185-191.
• [34]Cooper SJ, Trinklein ND, Anton ED, Nguyen L, Myers RM: Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome. Genome Res 2006, 16:1-10.
• [35]Tsuritani K, Irie T, Yamashita R, Sakakibara Y, Wakaguri H, Kanai A, Mizushima-Sugano J, Sugano S, Nakai K, Suzuki Y: Distinct class of putative “non-conserved” promoters in humans: comparative studies of alternative promoters of human and mouse genes. Genome Res 2007, 17:1005-1014.