| MicroRNAs (miRNAs) are a relatively recently identified class of regulatory noncoding RNAs, consisting of nearly 22 nucleotides in length. They play an important role in regulating gene expression by base-pairing to the complementary sites on the target mRNAs, thus blocking the translation or triggering the degradation of the target mRNAs. Since the discovery of the first miRNA (lin-4) in C. elegans, thousands of miRNAs have been identified experimentally or computationally from a variety of species. MiRNAs are currently estimated to comprise 1-5% of animal genes and collectively regulate up to 30% of genes, making them one of the most abundant classes of regulators. Although the number of known miRNAs is continuously increasing, information regarding their precise cellular function remains limited. One of the main challenges in understanding the functions of miRNAs is to identify the genuine target genes of miRNAs. However, lack of reliable and specific methods for biological target validation hampers the full understanding of the mechanisms by which miRNAs execute their functions. Only a few miRNAs have thus far been assigned target mRNAs, and the conventional methodologies are still labor intensive. Therefore, novel approaches for target identification are required to overcome this limitation.Here we show an efficient way to identify miRNA target genes by screening alterations in global mRNA levels following changes in miRNA levels. In this study, we used mRNA microarrays to measure global mRNA expression in three cell lines with increased or decreased levels of miR-16 and performed bioinformatics analysis based on multiple target prediction algorithms. For further investigation among the predicted miR-16 target genes, we selected genes that show an expression pattern opposite to that of miR-16. One of the candidate target genes that may interact with miR-16, ADP ribosylation factor-like protein 2 (ARL2), was further investigated. First, ARL2 was deduced to be an ideal miR-16 target by computational predictions. Second, ARL2 mRNA and protein levels were significantly abolished by treatment with miR-16 precursors, whereas a miR-16 inhibitor increased ARL2 mRNA and protein levels. Third, a luciferase reporter assay confirmed that miR-16 directly recognizes the 3’untranslated region (3’-UTR) of ARL2. Finally, we showed that miR-16 could regulate proliferation and induce a significant G0/G1 cell cycle arrest, which was due at least in part, to the downregulation of ARL2. In summary, the present study suggests that integrating global mRNA profiling and bioinformatics tools may provide the basis for further investigation of the potential targets of a given miRNA. These results also illustrate a novel function of miR-16 targeting ARL2 in modulating proliferation and cell cycle progression.UCP2 and UCP3, two novel uncoupling proteins, are important regulators of energy expenditure and thermogenesis in various organisms. The striking disparity between UCP2 mRNA and protein levels in muscle tissues prompted initial speculation that microRNAs are implicated in the regulatory pathway of UCP2. We found, for the first time, that the repression of UCP2 expression in cardiac and skeletal muscle resulted from its targeting by a muscle-specific microRNA, miR-133a. Moreover, our findings illustrate a novel function of UCP2 as a brake for muscle development. We also show that MyoD can remove the braking role of UCP2 via direct up-regulation of miR-133a during myogenic differentiation. Taken together, our current work delineates a novel regulatory network employing MyoD, microRNA, and uncoupling proteins to fine-tune the balance between muscle differentiation and proliferation during myogenesis. |
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