Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Dec 1;368(Pt 2):621–631. doi: 10.1042/BJ20020918

Cloning and molecular expression analysis of large and small lecithin:retinol acyltransferase mRNAs in the liver and other tissues of adult rats.

Reza Zolfaghari 1, Yuanping Wang 1, Qiuyan Chen 1, Anne Sancher 1, A Catharine Ross 1
PMCID: PMC1223011  PMID: 12201819

Abstract

Retinyl ester, the most abundant form of vitamin A (retinol), is synthesized by the enzyme lecithin:retinol acyltransferase (LRAT). Previously, we cloned a 2.5 kb LRAT cDNA from rodent liver which codes for functional LRAT activity. However, Northern blots of tissues probed with the 2.5 kb cDNA revealed the presence of a larger transcript of approximately 5 kb as well as several smaller transcripts. To elucidate the nature of the large LRAT transcript, a high-molecular-mass adrenal gland cDNA library was screened. Two similar clones of 3962 and 3187 nt were identified which appeared to be part of the 3'-untranslated region (UTR) of a 5358 nt LRAT mRNA. The 5.3 kb cDNA was then amplified from liver by reverse transcriptase PCR (RT-PCR) and demonstrated to encode functional LRAT activity. The 3'-UTR of the 5.3 kb cDNA contains several AAUAAA polyadenylation signals. Analysis of the 3' ends of LRAT mRNA transcripts from liver, intestine and testis showed the usage of both canonical and non-canonical polyadenylation signals. To further analyse the LRAT mRNAs expressed in vivo, Northern blot analysis was performed using four probes spanning sections from the 5' end to the distal 3' end of the 5.3 kb LRAT cDNA. The results show that the major 5.3 kb transcript uses the canonical signal AAUAAA located at nt 5308, and the major short transcript of approximately 1.5 kb uses the non-canonical signal AUUAAA located at nt 1330. The 5.3 kb LRAT transcript was predominant in the liver of retinoic acid-repleted vitamin A-deficient rats, coincident with increased quantitative expression of LRAT mRNA and enzyme activity. The differential usage of these polyadenylation signals can explain the presence of multiple LRAT mRNA transcripts which are expressed in different tissue-specific patterns.

Full Text

The Full Text of this article is available as a PDF (388.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andreola F., Giandomenico V., Spero R., De Luca L. M. Expression of a smaller lecithin:retinol acyl transferase transcript and reduced retinol esterification in MCF-7 cells. Biochem Biophys Res Commun. 2000 Dec 29;279(3):920–924. doi: 10.1006/bbrc.2000.3995. [DOI] [PubMed] [Google Scholar]
  2. Beaudoing E., Freier S., Wyatt J. R., Claverie J. M., Gautheret D. Patterns of variant polyadenylation signal usage in human genes. Genome Res. 2000 Jul;10(7):1001–1010. doi: 10.1101/gr.10.7.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen C. Y., Shyu A. B. AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem Sci. 1995 Nov;20(11):465–470. doi: 10.1016/s0968-0004(00)89102-1. [DOI] [PubMed] [Google Scholar]
  4. Conne B., Stutz A., Vassalli J. D. The 3' untranslated region of messenger RNA: A molecular 'hotspot' for pathology? Nat Med. 2000 Jun;6(6):637–641. doi: 10.1038/76211. [DOI] [PubMed] [Google Scholar]
  5. Edwalds-Gilbert G., Veraldi K. L., Milcarek C. Alternative poly(A) site selection in complex transcription units: means to an end? Nucleic Acids Res. 1997 Jul 1;25(13):2547–2561. doi: 10.1093/nar/25.13.2547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Guo X., Nanus D. M., Ruiz A., Rando R. R., Bok D., Gudas L. J. Reduced levels of retinyl esters and vitamin A in human renal cancers. Cancer Res. 2001 Mar 15;61(6):2774–2781. [PubMed] [Google Scholar]
  7. Guo X., Ruiz A., Rando R. R., Bok D., Gudas L. J. Esterification of all-trans-retinol in normal human epithelial cell strains and carcinoma lines from oral cavity, skin and breast: reduced expression of lecithin:retinol acyltransferase in carcinoma lines. Carcinogenesis. 2000 Nov;21(11):1925–1933. doi: 10.1093/carcin/21.11.1925. [DOI] [PubMed] [Google Scholar]
  8. MacDonald Clinton C., Redondo José-Luis. Reexamining the polyadenylation signal: were we wrong about AAUAAA? Mol Cell Endocrinol. 2002 Apr 25;190(1-2):1–8. doi: 10.1016/s0303-7207(02)00044-8. [DOI] [PubMed] [Google Scholar]
  9. Matsuura T., Ross A. C. Regulation of hepatic lecithin: retinol acyltransferase activity by retinoic acid. Arch Biochem Biophys. 1993 Mar;301(2):221–227. doi: 10.1006/abbi.1993.1137. [DOI] [PubMed] [Google Scholar]
  10. Matsuura T., Zhao Z., Ross A. C. N-(4-hydroxyphenyl)-retinamide increases lecithin:retinol acyltransferase activity in rat liver. J Nutr. 1996 Oct;126(10):2474–2480. doi: 10.1093/jn/126.10.2474. [DOI] [PubMed] [Google Scholar]
  11. Mitchell P., Tollervey D. mRNA stability in eukaryotes. Curr Opin Genet Dev. 2000 Apr;10(2):193–198. doi: 10.1016/s0959-437x(00)00063-0. [DOI] [PubMed] [Google Scholar]
  12. Ruiz A., Kuehn M. H., Andorf J. L., Stone E., Hageman G. S., Bok D. Genomic organization and mutation analysis of the gene encoding lecithin retinol acyltransferase in human retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2001 Jan;42(1):31–37. [PubMed] [Google Scholar]
  13. Ruiz A., Winston A., Lim Y. H., Gilbert B. A., Rando R. R., Bok D. Molecular and biochemical characterization of lecithin retinol acyltransferase. J Biol Chem. 1999 Feb 5;274(6):3834–3841. doi: 10.1074/jbc.274.6.3834. [DOI] [PubMed] [Google Scholar]
  14. Russo D. A., Petryk A., August C. S. Telomerase activity and phenotypic characterization in harvested bone marrow from a child with a germline cell cancer. Transplant Proc. 1997 Jun;29(4):2002–2002. doi: 10.1016/s0041-1345(97)00204-2. [DOI] [PubMed] [Google Scholar]
  15. Wang Yuanping, Zolfaghari Reza, Ross A. Catharine. Cloning of rat cytochrome P450RAI (CYP26) cDNA and regulation of its gene expression by all-trans-retinoic acid in vivo. Arch Biochem Biophys. 2002 May 15;401(2):235–243. doi: 10.1016/S0003-9861(02)00043-7. [DOI] [PubMed] [Google Scholar]
  16. Zolfaghari R., Chen X., Fisher E. A. Simple method for extracting RNA from cultured cells and tissue with guanidine salts. Clin Chem. 1993 Jul;39(7):1408–1411. [PubMed] [Google Scholar]
  17. Zolfaghari R., Ross A. C. Effect of vitamin A deficiency and retinoic acid repletion on intestinal and hepatic apolipoprotein A-I mRNA levels of adult rats. J Lipid Res. 1994 Nov;35(11):1985–1992. [PubMed] [Google Scholar]
  18. Zolfaghari R., Ross A. C. Lecithin:retinol acyltransferase from mouse and rat liver. CDNA cloning and liver-specific regulation by dietary vitamin a and retinoic acid. J Lipid Res. 2000 Dec;41(12):2024–2034. [PubMed] [Google Scholar]
  19. Zolfaghari Reza, Ross A. Catharine. Lecithin:retinol acyltransferase expression is regulated by dietary vitamin A and exogenous retinoic acid in the lung of adult rats. J Nutr. 2002 Jun;132(6):1160–1164. doi: 10.1093/jn/132.6.1160. [DOI] [PubMed] [Google Scholar]
  20. el Mansouri S., Tod M., Leclerq M., Petitjean O., Perret G., Porthault M. Time- and dose-dependent kinetics of all-trans-retinoic acid in rats after oral or intravenous administration(s). Drug Metab Dispos. 1995 Feb;23(2):227–231. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES