Characterization of a novel isoform of the human ORMDL3 gene.
The locus on chromosome 17q21, that encompasses the orosomucoid 1-like protein 3 (ORMDL3) gene, was considered a susceptibility locus associated to asthma, ulcerative colitis and ankylosing spondylitis, and polymorphisms within this locus were thought to be associated to an increased expression of the ORMDL3 gene. Several phosphorylation sites in the N-terminal regions of Orm proteins played crucial roles in the course of sphingolipid synthesis. To provide insight into our understanding of the expression of the ORMDL3 gene, we isolated and characterized a splicing isoform of ORMDL3, ORMDL3 V1, from Hela cells by 5'and 3'-rapid amplification of cDNA end analysis (RACE) and RT-PCR. ORMDL3 V1 skipped the second exon of the wild-type ORMDL3 gene. The predicted protein sequences of this isoform lacked 59 amino acids in the N-terminus of the wild-type ORMDL3 protein. RT-PCR assay showed that the mRNA levels of ORMDL3 V1 were higher in leukocytes, spleen, thymus, and Hela cells, lower in liver, brain, colon, lung, kidney, ovary, and testis. No mRNA expression was found in pancreas, heart, placenta, skeletal muscle, prostate, and small intestine. ORMDL3 V1 open reading frame was subcloned into pEGFP-C1 vector and it was found that the protein synthesis had been followed in transfected Hela cells. Western blot analysis detected a ∼38 kDa EGFP-ORMDL3 V1 fusion protein. Fluorescence microscopy demonstrated that both ORMDL3 V1 and ORMDL3 were almost exclusively expressed and localized in the cytoplasm of HEK293 cells. This study reveals the presence of a novel ORMDL3 splicing isoform, ORMDL3 V1 in human.
Department of Pediatrics, the First Affiliated Hospital, Nanjing Medical University, 300 Guang Zhou Road, Nanjing, Jiangsu Province 210029, China.
Rui
R
Jin
Wen-Xiao
WX
Yuan
Hua-Guo
HG
Xu
Wei
W
Ren
Li-Li
LL
Zhuang
Guo-Ping
GP
Zhou
D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D018076:DNA, Complementary; D008565:Membrane Proteins; C464399:ORMDL3 protein, human; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins