A study of miRNAs targets prediction and experimental validation

Protein & Cell, Dec 2010

microRNAs (miRNAs) are 20–24 nucleotide (nt) RNAs that regulate eukaryotic gene expression post-transcriptionally by the degradation or translational inhibition of their target messenger RNAs (mRNAs). To identify miRNA target genes will help a lot by understanding their biological functions. Sophisticated computational approaches for miRNA target prediction, and effective biological techniques for validating these targets now play a central role in elucidating their functions. Owing to the imperfect complementarity of animal miRNAs with their targets, it is difficult to judge the accuracy of the prediction. Complexity of regulation by miRNA-mediated targets at protein and mRNAs levels has made it more challenging to identify the targets. To date, only a few miRNAs targets are confirmed. In this article, we review the methods of miRNA target prediction and the experimental validation for their corresponding mRNA targets in animals.

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A study of miRNAs targets prediction and experimental validation

Protein Cell A study of miRNAs targets prediction and experimental validation Yong Huang 0 2 Quan Zou 1 Haitai Song 0 2 Fei Song 0 2 Ligang Wang 0 2 Guozheng Zhang 0 2 Xingjia Shen 0 2 0 The Key Laboratory of Silkworm and Mulberry genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences , Zhejiang 212018 , China 1 School of information Science and Technology of Xiamen University , Xiamen 361005 , China 2 Jiang Su University of Science and Technology , Zhenjiang 212018 , China microRNAs (miRNAs) are 20-24 nucleotide (nt) RNAs that regulate eukaryotic gene expression post-transcriptionally by the degradation or translational inhibition of their target messenger RNAs (mRNAs). To identify miRNA target genes will help a lot by understanding their biological functions. Sophisticated computational approaches for miRNA target prediction, and effective biological techniques for validating these targets now play a central role in elucidating their functions. Owing to the imperfect complementarity of animal miRNAs with their targets, it is difficult to judge the accuracy of the prediction. Complexity of regulation by miRNA-mediated targets at protein and mRNAs levels has made it more challenging to identify the targets. To date, only a few miRNAs targets are confirmed. In this article, we review the methods of miRNA target prediction and the experimental validation for their corresponding mRNA targets in animals. microRNA; computational prediction; target; experimental validation INTRODUCTION The miRNAs are a widely distributed class of small noncoding RNAs that play an integral role in gene regulation (Elbashir et al., 2001; Hutvágner and Zamore, 2002) . miRNA biogenesis pathway in animals can be divided into two steps (Fig.1). Initially, miRNAs are transcribed by RNA polymerase II as primary miRNAs (pri-miRNAs) with hundreds to thousands of nucleotides in length (Cai et al., 2004; Lee et al., 2004; Trujillo et al., 2010) . Ribonuclease III (RNase III) enzyme Drosha cleaves the flanks of pri-miRNAs to liberate ~70 nucleotide stem-loop structures, called precursor miRNAs (pre-miRNAs). Pre-miRNA hairpins are exported from the nucleus by Exportin-5 (Lee et al., 2003; Engels and Hutvagner, 2006; Flynt and Lai, 2008) . In the cytoplasm, the pre-miRNAs are processed into ~22 nucleotide duplex miRNAs (miR/miR*) by the RNase III enzyme Dicer (Kim, 2004; Lund et al., 2004; Engels and Hutvagner, 2006) . Next, one strand of the miRNA duplex is loaded into the RISC (RNA-induced silencing complex) to bind the mRNA target. If the complementarities between the 3′-UTR mRNA and the miRNA are extensive, the target mRNA is degraded; whereas, if the complementarities are partial, the translation of the target mRNA is repressed (Brennecke et al., 2005) . Recent studies have shown that many miRNAs are involved in a variety of biological processes, such as transcriptional gene regulatory network, developmental timing, neuronal synapses formation, cell proliferation, cell death, viral infection, differentiation and tumor metastasis (Sarnow et al., 2006; Hwang and Mendell, 2007; Ma et al., 2007; Bartel, 2009; Nachmani et al., 2009; Xiao and Rajewsky, 2009) . Currently, more than 10,000 miRNAs have been identified in the miRBase database (http://www. mirbase.org/). Thus, the development of precise and fast assays for miRNA target identification and verification will play a significant role in the study of miRNA functions and the biological processes in which they are involved. Several effective algorithms have been developed for the prediction of miRNAs targets in animals. In this review, we summarize the prediction methods of miRNAs targets and the experimental approaches that have been described for identification of their targets. For miRNAs in animals, the target prediction is more complex because few miRNAs are perfectly complementary to their targets. In the following only animal miRNAs are considered. Principles of miRNA target recognition The function of a miRNA is ultimately defined by its targets and the effects it has on their expression. Although the detailed target recognition mechanism is still elusive, the consensus suggests that the base pairing of miRNA with its target mRNA is the key. Differences in target complementarities and target location within the mRNA could be related to the silencing mechanism used. The prediction criteria include the following: 1) The miRNA sequence is complementary to the 3′-UTR sequence of potential target mRNAs. Especially, the strong binding of the 5′ end (the first eight base pairs) of the mature miRNA to the 3′-UTR sequence is very important for targeting, whereas the G:U wobble pairing reduces the silencing efficiency (Brodersen and Voinnet, 2009) . For example, there are three types of target sites: 5′-dominant canonical, 5′-dominant seed only and 3′-compensatory (Fig. 2). They differ in the level of comp (...truncated)


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Yong Huang, Quan Zou, Haitai Song, Fei Song, Ligang Wang, Guozheng Zhang, Xingjia Shen. A study of miRNAs targets prediction and experimental validation, Protein & Cell, 2010, pp. 979-986, Volume 1, Issue 11, DOI: 10.1007/s13238-010-0129-4