MicroRNA Mechanisms of Action: What have We Learned from Mice?

MicroRNAs (miRNAs) are endogenously encoded single-stranded RNAs of about 22 nucleotides (nts) in length that play essential roles in a large variety of physiological processes in animals and plants (Ambros, 2004; Bushati and Cohen, 2007). Mature miRNAs are integrated into the RNA-induced silencing complex (RISC), whose core component is one of the Argonaute family proteins. MiRNAs then direct RISCs to target mRNAs, which are recognized through partial sequence complementarity. Bioinformatic prediction and experimental target gene identification have shown that a miRNA binds mRNAs of hundreds of protein coding genes, which often span a broad spectrum of functional categories (Bartel, 2009; Chi et al., 2009; Hafner et al., 2010). The functional consequence of miRNA-target mRNA interaction and the mechanism of miRNA action have been under intensive investigation and remain a matter of hot debate. It was initially thought that miRNAs repress the protein output of a small number of target genes without significantly affecting their mRNA levels in animals (Lee et al., 1993; Wightman et al., 1993). Subsequent genetic studies in C. elegans and zebrafish showed that miRNAs promote the degradation of their target mRNAs (Bagga et al., 2005; Giraldez et al., 2006). Later, a series of genome-wide studies of in vitro cultured mammalian cell lines transiently transfected with chemically synthesized miRNA mimics led to the conclusion that the predominant functional consequence of miRNA action is target mRNA degradation (Guo et al., 2010). A follow-up study employing temporal dissection of zebrafish development seems to reconcile these two opposite observations by revealing that translational repression precedes target mRNA decay, and suggesting that the immediate outcome of miRNA-target mRNA interaction is translation inhibition but mRNA degradation can follow (Bazzini et al., 2012). Similarly, re-analysis of the previous datasets from cultured cell lines transiently transfected with synthetic miRNA mimics also found that translation repression precedes mRNA degradation (Larsson and Nadon, 2013).

MicroRNAs (miRNAs) are endogenously encoded single-stranded RNAs of about 22 nucleotides (nts) in length that play essential roles in a large variety of physiological processes in animals and plants (Ambros, 2004;Bushati and Cohen, 2007). Mature miRNAs are integrated into the RNAinduced silencing complex (RISC), whose core component is one of the Argonaute family proteins. MiRNAs then direct RISCs to target mRNAs, which are recognized through partial sequence complementarity. Bioinformatic prediction and experimental target gene identification have shown that a miRNA binds mRNAs of hundreds of protein coding genes, which often span a broad spectrum of functional categories (Bartel, 2009;Chi et al., 2009;Hafner et al., 2010). The functional consequence of miRNA-target mRNA interaction and the mechanism of miRNA action have been under intensive investigation and remain a matter of hot debate. It was initially thought that miRNAs repress the protein output of a small number of target genes without significantly affecting their mRNA levels in animals (Lee et al., 1993;Wightman et al., 1993). Subsequent genetic studies in C. elegans and zebrafish showed that miRNAs promote the degradation of their target mRNAs (Bagga et al., 2005;Giraldez et al., 2006). Later, a series of genome-wide studies of in vitro cultured mammalian cell lines transiently transfected with chemically synthesized miRNA mimics led to the conclusion that the predominant functional consequence of miRNA action is target mRNA degradation (Guo et al., 2010). A follow-up study employing temporal dissection of zebrafish development seems to reconcile these two opposite observations by revealing that translational repression precedes target mRNA decay, and suggesting that the immediate outcome of miRNAtarget mRNA interaction is translation inhibition but mRNA degradation can follow (Bazzini et al., 2012). Similarly, re-analysis of the previous datasets from cultured cell lines transiently transfected with synthetic miRNA mimics also found that translation repression precedes mRNA degradation (Larsson and Nadon, 2013). However, the model miRNA used in the aforementioned zebrafish study, miR-430, is unique in that its expression is rapidly induced and reaches millions of copies per cell in a few hours after fertilization. This expression level of miR-430 is at least 10 times more than all mature miRNAs combined in a mammalian cell, and serves the single purpose of degrading its target genes, maternal mRNAs, at the maternal-zygotic transition (Giraldez et al., 2006). Mammalian cells often express 100-200 different species of miRNAs (Kuchen et al., 2010), with a total amount of 1-2 × 10 5 copies of mature miRNAs in a cell (Calabrese et al., 2007;Janas et al., 2012). The most abundant miRNAs are often expressed at the level of ∼2 × 10 4 copies per cell (Neilson et al., 2007;Kuchen et al., 2010). As an extreme example, miR-122 is expressed at the estimated level of 5 × 10 4 copies per cell in hepatocytes (Chang et al., 2004;Jopling et al., 2005). This is still about 20 times lower than the million-copy-per-cell expression level of miR-430 in zebrafish embryos. Considering that the estimated copy number of Argonaute proteins in a mammalian cell is of the same order of magnitude as the total amount of mature miRNAs (1.5 × 10 4 -1.7 × 10 5 ; Janas et al., 2012;Wang et al., 2012), the million-copy-per-cell expression level of miR-430 is unlikely to be physiologically relevant in mammalian cells. Therefore, the in vivo mechanism of action of mammalian miRNAs remains to be a central question in the field of miRNA research.
In contrast to these desperate efforts to search for a unified model of miRNA mechanism of action, studies of individual functional targets in primary cells or tissues from miRNA mutant mice are painting a rather different picture. Depending on miRNAs, target genes, and cellular contexts, the outcome of miRNA-target mRNA interactions could be predominantly translation repression or mRNA degradation, or a mixture of both. This heterogeneity in miRNA mechanisms of action has been increasingly recognized as more and more miRNA mutant mice are generated and analyzed (Olive et al., 2015), but a comprehensive review of relevant literature is still missing.
in mature miRNAs and target mRNAs determine the mechanism of miRNA action. Future investigation is warranted to identify these cis-elements, if they exist at all.
From a practical standpoint, measuring target gene protein levels is preferred to mRNA levels for the purpose of studying the effect of a miRNA on its target genes. Even for target genes predominantly regulated by mRNA degradation, the miRNA effect can still be captured by measuring their protein abundance. In the same vein, translatome analysis is more appropriate for measuring the global effect of a miRNA on its target genes, while transcriptome analysis often failed to identify any significant effect of miRNA deletion on its target genes, despite the obvious functional consequences in mutant mice (Matkovich et al., 2010;Boldin et al., 2011;Jiang et al., 2011;Agudo et al., 2014;Sullivan et al., 2015;Yuan et al., 2015). In the broader context of gene expression regulation, accumulating evidence shows that proteome and transcriptome are not sufficiently correlated to act as proxies for each other (Payne, 2015). miRNAmediated translation regulation may play an important role in the de-coupling of translatome from transcriptome. We speculate that miRNAs emerged during evolution to increase the complexity of gene regulation, thereby contributing to the diversity of organisms.

ACKNOWLEDGMENTS
We thank Dr. Li-Fan Lu (UCSD) for discussion and Jovan Shepherd for critical reading of this manuscript. This study is supported by the PEW Charitable Trusts, Cancer Research Institute, Lupus Research Institute, and National Institute of Health (R01AI087634, R01AI089854, and R56 AI110403 to CX). CX is a Pew Scholar in Biomedical Sciences.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fgene. 2015.00328