Expression of miR-33 from an SREBF2 Intron Targets the FTO Gene in the Chicken

Fang Shao,Xingguo Wang,Honglin Jiang,Jianfeng Yu,Bin Zhu,Zhiliang Gu,Andre Van Wijnen

doi:10.1371/journal.pone.0091236

Abstract

The sterol regulatory element binding transcription factor 2 (SREBF2) gene encodes a transcription factor that activates the expression of many genes involved in the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids. Through bioinformatics, we found that intron 16 of the chicken SREBF2 gene might encode the chicken miR-33. Using quantitative RT-PCR, we detected the expression of miR-33 in a variety of chicken tissues including skeletal muscle, adipose tissue, and liver. Three hundred and seventy eight genes were predicted to be potential targets of miR-33 in chickens via miRNA target prediction programs “miRanda” and “TargetScan”. Among these targets, the gene FTO (fat mass and obesity associated) encodes a Fe(II)- and 2-oxoglutarate-dependent nucleic acid demethylase that regulates lipid metabolism, and the possibility that its expression is negatively regulated by miR-33 in the chicken liver was therefore further studied. Co-transfection and dual-luciferase reporter assays showed that the expression of luciferase reporter gene linked to the 3′-untranslated region (3′UTR) of the chicken FTO mRNA was down-regulated by overexpression of the chicken miR-33 in the C2C12 cells (P<0.05). Furthermore, this down-regulation was completely abolished when the predicted miR-33 target site in the FTO 3′UTR was mutated. In contrast, the expression of FTO mRNA in the primary chicken hepatocytes was up-regulated after transfection with the miR-33 inhibitor LNA-anti-miR-33. Using quantitative RT-PCR, we also found that the expression of miR-33 was increased in the chicken liver from day 0 to day 49 of age, whereas that of the FTO mRNA was decreased during the same age period. These data together suggest that miR-33 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of the FTO gene.

Highlights

IntroductionIn addition to classical transcription factors, a new class of noncoding RNAs termed microRNAs (miRNAs) has emerged as critical regulators of gene expression acting predominantly at the posttranscriptional level. miRNAs are single-stranded small RNA molecules, with the length of 18,25 nucleotides (nt)
In addition to classical transcription factors, a new class of noncoding RNAs termed microRNAs has emerged as critical regulators of gene expression acting predominantly at the posttranscriptional level. miRNAs are single-stranded small RNA molecules, with the length of 18,25 nucleotides. They bind to the 39-untranslated regions (39UTR) of mRNA transcripts to reduce the translation of these transcripts or to cause their degradation [1]
MicroRNA-33 is transcribed from an intronic region within the sterol response element binding transcription factor 2 (SREBF2), called sterol response element binding protein-2 gene [5], which directly activates the expression of more than 30 genes involved in the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids [6,7]. miR-33 is expressed in numerous mammalian cell types and tissues [8,9]

Summary

Introduction

In addition to classical transcription factors, a new class of noncoding RNAs termed microRNAs (miRNAs) has emerged as critical regulators of gene expression acting predominantly at the posttranscriptional level. miRNAs are single-stranded small RNA molecules, with the length of 18,25 nucleotides (nt). MiRNAs are single-stranded small RNA molecules, with the length of 18,25 nucleotides (nt) They bind to the 39-untranslated regions (39UTR) of mRNA transcripts to reduce the translation of these transcripts or to cause their degradation [1]. Research by multiple groups has shown that miR-33 analogs regulate cholesterol and fatty acid metabolism in mammalian systems, corresponding with the function of its host gene [10,11]. In addition to regulating cholesterol transport, high-density lipoprotein metabolism and fatty acid b-oxidation, miR-33 was recently reported to regulate cell cycle progression and cellular proliferation [12], inflammatory response [13] and insulin signaling [14]

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