Supplementary site interactions are critical for the regulation of microsomal triglyceride transfer protein by microRNA-30c

James Soh,M Hussain

doi:10.1186/1743-7075-10-56

Abstract

Microsomal triglyceride transfer protein (MTTP) is an essential chaperone that assists in the assembly of apolipoprotein B-containing lipoproteins to transport lipids. We have shown that microRNA (miR)-30c regulates MTTP expression but other members of the same family do not. Further, we showed that interactions between miR-30c seed sequence and the 3΄-untranslated region (UTR) of the MTTP mRNA are critical for this regulation. The same seed sequence is shared by all the members of the miR-30 family. Therefore, it is unclear why only miR-30c regulates MTTP expression. Bioinformatics analysis revealed that, miR-30c interacts with MTTP mRNA involving supplementary site, besides seed sequence, forming an intervening loop. Here, we evaluated the importance of the supplementary site and the size of the intervening loop in miR-30c/MTTP mRNA interactions by cloning MTTP 3΄-UTR at the end of the luciferase gene and subjecting it to site-directed mutagenesis. Reducing the number of base pairs at the supplementary site abolished the ability of miR-30c to reduce luciferase activity. However, increasing the number of base pairs at the supplementary site, seed sequence or in the intervening loop enhanced the efficacy of miR-30c in reducing luciferase activity. These studies demonstrated that the supplementary site of miR-30c is, but the intervening loop is not, critical for binding to the MTTP mRNA. To our knowledge, this is the first demonstration that miRs might require both seed and supplementary interactions to regulate target mRNA specificity. Further, this study suggests that more potent miR-30c mimics could be synthesized by increasing base pairing in the loop region.

Highlights

The lipoprotein secretion pathway is controlled by two critical factors: Microsomal triglyceride transfer protein (MTTP) and apolipoprotein B [1]
Comparative analysis of the miR-30 family members and their possible interactions with MTTP mRNA revealed that these two molecules could form complementary base pairing involving disparate “seed” and “supplementary” sequences resulting in the formation of an intervening asymmetric loop where 8 residues in miR-30c and 6 residues in MTTP mRNA do not form base pairs (Figure 1)
We have previously shown that miR-30c lowers both MTTP mRNA and protein levels, but other miR-30 family members do not affect MTTP levels [13]

Summary

Introduction

The lipoprotein secretion pathway is controlled by two critical factors: MTTP and apolipoprotein B (apoB) [1]. The significance of MTTP was initially revealed in a condition called abetalipoproteinemia [2] These patients have null mutations in the MTTP gene and exhibit virtually no plasma apoB lipoproteins [3,4]. This discovery highlighted the importance of MTTP in the lipoprotein assembly and secretion pathway and suggested the possibility that its inhibition might be useful in reducing plasma lipids. Several chemical inhibitors of MTTP have been developed These antagonists successfully block MTTP activity and lower plasma lipids. They cause hepatic steatosis and increase plasma transaminases [5,6]. Intestinespecific MTTP antagonists have been designed to circumvent unwanted side effects associated with hepatic MTTP inhibition [9]

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