Cloning, gene organization and identification of an alternative splicing process in lecithin:retinol acyltransferase cDNA from human liver

Abstract

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.

Fig. 1

Cloning of full-length LRAT mRNA from human liver. (A, B) Identification of the 5′-end of the LRAT mRNA. Ethidium bromide-stained agarose gel after electrophoresis of the PCR products (A) and nested PCR products (B) of the 5′-end of LRAT mRNA as cloned by the GeneRacer kit (see text for details). M, low-mass DNA marker. (C) Amplification of full-length LRAT mRNA from human liver. Ethidium bromide-stained agarose gel of the LRAT RT-PCR product amplified from human liver poly[A]⁺ RNA. Either of two sense primers (primers 10 and 11) with an antisense primer (primer 12) was designed from the 5′- and 3′-ends of the LRAT mRNA (lane 3, primer pair 10 and 12; lane 4, primer pair 11 and 12, see Table 1 for primer identification), and used for RT-PCR as described in Materials and methods. The LRAT amplicons from human liver had the expected size of about 4.8–4.9 kb. Lane 1, negative control; lane 2, high-mass DNA marker.

Fig. 2

Organization of human LRAT gene. Schematic organization of LRAT gene relative to the full-length and spliced forms of the LRAT mRNA transcripts. The empty box represents the UTR and the filled box indicates the coding region. The box and the thick line in the LRAT gene represent exon and intron, respectively. The solid arrows indicate the position of the primers used to clone and analyze the LRAT cDNA and the numbers on the top and bottom of the LRAT transcript represent the sense and antisense primers (see Table 1), respectively.

Fig. 3

Identification of the full-length and spliced forms of the LRAT mRNA transcripts in human liver and testis. (A, B) The presence and absence of the polynucleotide corresponding to the first intron of the LRAT gene in the LRAT mRNA transcripts. Ethidium bromide-stained agarose gel electrophoresis of RT-PCR products of human liver poly[A]⁺ RNA (A) and of PCR products from the DNA template extracted from the gel shown in Fig. 1C (B). Primer pairs used are as follows: lane 1, primers 8 and 3; lane 2, primers 8 and 4; lane 3; primers 8 and 14; lane 4, primers 8 and 13; lane 5, primers 8 and 9 (see Table 1 for position of the primers). M, low-mass DNA marker. (C) Cloning and analysis of the full-length and spliced form of the human liver LRAT mRNA transcripts. The DNA from the gel shown in lane 3 of Fig. 1C was cut, extracted and cloned by TA cloning as described in Materials and methods. Two clones were picked up for digestion with SacII and NotI enzymes. Lane 1, spliced form clone; lane 2, the intron inclusion form clone. M1 and M2 are high- and low-mass DNA markers, respectively. (D) Expression of LRAT mRNA in individual human liver samples. Ethidium bromide-stained agarose gel electrophoresis of RT-PCR products of total RNA from liver samples, classified as normal, of five individuals. Total RNA from individual samples was extracted and treated with DNase to remove any genomic DNA followed by RT-PCR analysis as described in detail in Materials and methods using primers number 8 and 9 (see Table 1 for sequence and position of the primers). M, low-mass DNA marker; C, negative control. (E) Expression of LRAT spliced forms in human testis. Ethidium bromide-stained agarose gel electrophoresis of RT-PCR products of human testis total RNA using the same sets of primers used in A and B.