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

Abstract

Retinyl ester, the most abundant form of vitamin A (retinol), is synthesized by the enzyme lecithin:retinol acyltransferase (LRAT). Previously, we cloned a 2.5kb LRAT cDNA from rodent liver which codes for functional LRAT activity. However, Northern blots of tissues probed with the 2.5kb cDNA revealed the presence of a larger transcript of 5kb 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 3187nt were identified which appeared to be part of the 3′-untranslated region (UTR) of a 5358nt LRAT mRNA. The 5.3kb cDNA was then amplified from liver by reverse transcriptase PCR (RT-PCR) and demonstrated to encode functional LRAT activity. The 3′-UTR of the 5.3kb 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 5.3kb LRAT cDNA. The results show that the major 5.3kb transcript uses the canonical signal AAUAAA located at nt 5308, and the major short transcript of 1.5kb uses the non-canonical signal AUUAAA located at nt 1330. The 5.3kb 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.