Cloning, gene organization and identification of an alternative splicing process in lecithin:retinol acyltransferase cDNA from human liver.
Lecithin:retinol acyltransferase (LRAT) catalyzes the synthesis of retinyl esters in many tissues and is crucial for the transport and intracellular storage of vitamin A. LRAT expression is highly regulated in the liver. In this study, we have cloned and sequenced the full-length LRAT mRNA from human liver and identified its 5'- and 3'-ends. Full-length LRAT mRNA comprises 5023 nt with a predicted ORF of 230 amino acids, a short 5'UTR, and a relatively long 3'UTR of 4 kb containing several polyadenylation signals and AU-rich regions. Based on alignment of this mRNA with human genomic DNA in the GenBank database, the human LRAT gene spans about 9.1 kbp and consists of two exons and a relatively long 4-kbp intron. Further analysis of normal liver revealed a minor alternative splicing variant which lacks a 103 nt polynucleotide contained in the 5'UTR of the full-length LRAT transcript. This variant predicts that the LRAT gene is organized into three exons and two introns, as reported for LRAT cloned from retinal pigment epithelium (RPE) cells. These two LRAT mRNA variants are also present in testis, which is known to express LRAT and contain retinyl esters. Major and minor transcription start sites for human liver LRAT mRNA were identified and the sequence of the upstream proximal promoter region was retrieved from the GenBank database and physically analyzed for the presence of putative cis-acting elements essential for basal transcription. This region contains a TATA box, CCAAT box and Sp1 site, which are apparently conserved in mouse and rat LRAT genes. Our results provide evidence that multiple LRAT mRNA transcripts, which are expressed in a tissue-specific manner, may result from several mechanisms including differential splicing of the 5'UTR region and the use of multiple polyadenylation signals in the 3'UTR.
Department of Nutritional Sciences, and Huck Life Science Institute, S-126 Henderson Building, The Pennsylvania State University, University Park, PA 16802, USA.
Reza
R
Zolfaghari
A Catharine
AC
Ross
D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000217:Acyltransferases / Q000235:genetics*; D017398:Alternative Splicing; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes / Q000235:genetics; D006801:Humans; D007438:Introns; D007527:Isoenzymes / Q000235:genetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D016385:TATA Box / Q000235:genetics; D013737:Testis / Q000201:enzymology / Q000378:metabolism
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D000217:Acyltransferases; C056942:lecithin-retinol acyltransferase