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Gene regulation by microRNAs

Richard W Carthew
The role of small RNAs as key regulators of mRNA turnover and translation has been well established. Recent advances indicate that the small RNAs termed microRNAs play important roles in animal development and physiology. Cellular activities such as proliferation, morphogenesis, apoptosis and differentiation are regulated by microRNAs. The expression of various genes are regulated by microRNAs, and several microRNAs act in reciprocal negative feedback loops with protein factors to control cell fate decisions that are triggered by signal transduction activity. These observations implicate small RNAs as important mediators of gene regulation in response to cellcell signaling. The mechanism by which microRNAs silence gene expression is post-transcriptional, possibly inuencing the stability, compartmentalization and translation of mRNAs. This mechanism is an efcient means to regulate production of a diverse range of proteins.
Addresses Department of Biochemistry, Molecular Biology and Cell Biology, 2205 Tech Drive, Northwestern University, Evanston, IL 60208, USA Corresponding author: Carthew, Richard W (rcarthew@northwestern.edu)

MicroRNA biogenesis and mechanism


The number of miRNA genes found in sequenced animal species corresponds to approximately 0.51.5% of the total number of genes in their genomes (http://www. sanger.ac.uk/Software/Rfam/mirna/index.shtml). The fruit y Drosophila melanogaster and the nematode Caenorhabditis elegans have, at the latest count, 78 and 114 miRNA genes, respectively. Humans have at least 326 miRNA genes. MicroRNAs are transcribed by RNA polymerase II, and the primary transcripts contain hairpinloop domains that fold back into duplex-like structures [1,2]. The hairpinloop region is cleaved in the nucleus by the RNase III enzyme Drosha in complex with a double-strand RNA-binding protein termed DGCR8 [36]. The resulting hairpinloop fragment is then exported from the nucleus to the cytoplasm, where it is cleaved by the RNase III enzyme Dicer in partnership with another RNA-binding protein called TRBP (transactivation-responsive RNA-binding protein) [79]. The fully mature miRNA accumulates as a single-stranded species comprising one arm of each miRNA hairpin precursor, and it incorporates into a ribonucleoprotein complex that carries out its function of silencing gene expression. Although the exact silencing mechanism is not known, it is clear that miRNAs use a mode of silencing related to that employed by siRNAs, which cleave mRNA transcripts. In a similar fashion to siRNAs, miRNAs inhibit a target mRNA by base-pairing to complementary sequences within the message. However, an animal miRNA typically makes imperfect base pair contacts with its target mRNA. Contacts usually occur within 30 untranslated regions (UTRs). The historical mechanistic view has been that miRNA-bound target mRNAs are not cleaved but, instead, are unable to generate encoded proteins, due to an effect on the elongation phase of protein synthesis [10]. Recently, this view has come into question with a bevy of discoveries suggesting that miRNAs might inhibit gene expression by other mechanisms. Human let-7 miRNA inhibits translation of reporter mRNAs through a (mRNA 50 ) cap-dependent mechanism that affects initiation; a similar effect was seen with an articial miRNA [11,12]. This discrepancy is further exacerbated by independent ndings that implicate miRNAs as destabilizers of imperfectly base-paired mRNAs. Human miRNAs miR-1 and miR-124 cause a reduction in the abundance of target transcripts [13]. Moreover, miR16 is complementary to AU-rich elements found in the 30 UTRs of some short-lived transcripts, and miR-16 is essential for the rapid turnover of these mRNAs [14]. Finally, both lin-4 and let-7 miRNAs in C. elegans
Current Opinion in Genetics & Development 2006, 16:203208

Current Opinion in Genetics & Development 2006, 16:203208 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by Susan Parkhurst and Toshio Tsukiyama Available online 28th February 2006 0959-437X/$ see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2006.02.012

Introduction
Although the role that proteins play in gene regulation is well understood, it has become clear that RNAs are also important gene regulatory factors. Small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), are components of an RNA-based mechanism of gene regulation found in eukaryotes. siRNAs are used throughout the Eukaryota to inhibit viruses and transposable elements. They also play a role in chromosome organization and in silencing the expression of proteincoding genes. The miRNA branch of RNA-based gene regulation is less widespread; miRNAs are found in plants and animals but are apparently absent in fungi. This review focuses upon recent advances in our understanding of miRNAs and their manifest functions in animal development and physiology.
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204 Chromosomes and expression mechanisms

destabilize target mRNAs even though they are imperfectly base-paired [15]. It is not yet clear whether these observed reductions in mRNA levels are a result of cleavage by a siRNA-like mechanism or whether the bound miRNAs enlist other degradation machineries. The data are further compounded by recent observations that miRNAs might repress gene expression by sequestering targeted mRNAs into P-/GW-bodies (processing bodies) [16,17]. These are cytoplasmic foci that contain non-translated mRNAs and exclude the translation machinery. Such target localization could potentially embody part, or all, of the underlying cause of repression. One simple model is that the interaction between miRNA and target results in transport of mRNAs to P-/ GW-bodies, where they are unavailable to the protein synthetic machinery but are subject to de-capping and degradation by resident nucleases. It is also conceivable that miRNAs act through multiple cooperative mechanisms to repress their targets. Consistent with this notion, other specic translation repression mechanisms have been observed to feed into P-/GW-bodies.

the expression of each effector miRNA in various tissues. This suggests that the transcripts that are downregulated in HeLa cells are biological targets of the same miRNAs expressed in human tissues. For each miRNA, approximately 100 transcripts were downregulated, suggesting a large miRNA:target ratio, in the same order as that predicted by computational methods. A second approach to nding genome-wide targets by experimental analysis used proteomics [22]. Unfertilized oocytes were harvested from mutant Drosophila females missing the dicer-1 gene, and thus depleted of properly processed miRNAs. The proteome from such oocytes was compared to the normal oocyte proteome, and 4% of the detected proteins were found to be downregulated when miRNAs are normally processed. Of the proteins identied by mass spectrometry, virtually all had predicted miRNA-binding sites within their 30 UTRs. The two genome-wide approaches have provided somewhat contradictory conclusions. The range and breadth of targets is noticeably restricted in the Drosophila oocyte in comparison with that in HeLa cells. A much smaller proportion of the genome appears to be repressed by y miRNAs compared with the proportion in sample human miRNAs tested. Moreover, most of the Drosophila targets are involved in some aspect of global protein metabolism, whereas the human targets have more diverse functions. The Drosophila targets contain primarily 30 compensatory binding sites, and most target transcripts are not reduced in abundance. These differences between Drosophila and human results are not yet fully explainable. It might reect an intrinsic difference in miRNA mechanisms between the two species, but more probably it reects the unusual features of late-stage oocytes in general. Oocytes shut off all RNA synthesis, and RNA metabolism is highly regulated, as is protein metabolism. Intriguingly, under certain stress conditions, mammalian cells use miRNAs to regulate global protein metabolism [23].

What are the targets of miRNA regulation?


A major question about miRNAs concerns the extent of their regulation of animal genomes. Computational methods have been helpful in estimating this value. When the 30 UTRs from sequenced mammalian genomes were aligned, sequences capable of forming 68 bp perfect duplexes with the 50 ends of many human miRNAs were identied [18]. Given the frequency of 30 UTR motifs with complementary miRNAs found, it was estimated that approximately 20 to 30% of all human genes are targets of miRNA regulation, and there is an average of 200 targets per miRNA [18,19]. Most binding sites are dominated by complementarity to the miRNA seed, with little or no support from pairing to the 30 end [20]. By contrast, other binding sites contain imperfect seed pairing and are compensated with strong 30 pairing [20,21]. These 30 compensatory sites are estimated to constitute 20% of all miRNA-binding sites. Bioinformatic studies have suggested that the extent of miRNA-based gene regulation has remained relatively stable throughout animal evolution, with 15 to 30% of animal genomes under direct regulation. A formidable task remains in testing these modeling predictions. Although small-scale experimental validation studies have conrmed the efcacy of these computational models, it has been more difcult to determine the genome-wide extent of miRNA control. Two approaches have been adopted with some success. One approach sought targets by transfecting tissue-specic miRNAs into HeLa cells and identifying which mRNAs were consequently reduced in abundance [13]. Of the transcripts that were repressed by each miRNA, a negative correlation was observed between their expression and
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Biological regulation by miRNAs: global approaches


If 30% of the coding genome is repressed by miRNAs, then the breadth of regulated biological processes might be enormous. To address this issue, several groups have investigated the consequences of eliminating miRNA maturation by examining the phenotypes of Dicer mutant animals. Surprisingly, they have revealed rather focused decits that suggest more specic roles for miRNAs. Knockout of mouse Dicer results in embryonic lethality, and conditional removal of Dicer from the embryonic limb causes extensive apoptosis [24]. However, patterning and differentiation of the mutant limb remains mostly normal. This strong dependence on Dicer for cell survival was also observed in the T-cell lineage of the hematopoietic system [25]. Loss of Dicer in zebrash results in abnormal
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Gene regulation by microRNAs Carthew 205

morphogenesis during gastrulation, neurogenesis, and somitogenesis; but, again, overall patterning of the body axis is normal [26]. Such tissue-specic roles for microRNAs are supported by the documented expression patterns of zebrash miRNAs; most miRNAs are expressed in highly tissue-specic patterns during segmentation and later stages [27]. A different role for miRNAs has been revealed in stem cells. One of the key characteristics of stem cells is their capacity to divide for long periods of time in an environment where most of the cells are quiescent. A crucial question in stem cell biology has been how stem cells escape division stop signals. It appears that miRNAs are required for division of germ line stem cells (GSCs) in Drosophila [28]. If GSCs are mutant for Dicer-1, then the frequency of GSC division drops by 80%, and many fewer eggs and sperm are produced. This cessation is not caused by a defect in GSC differentiation but, rather, by a defect in cell cycle control; mutant GSCs are blocked at the G1 S checkpoint as a result of upregulation of the cyclindependant kinase-inhibitor Dacapo. Binding sites for several miRNAs are located in the 30 UTR of Dacapo, suggesting that miRNAs are required for GSCs to bypass the normal G1S checkpoint by downregulating this inhibitor. The miRNA pathway might be part of a general mechanism that promotes stem cell proliferation, because Dicer mutant embryonic stem cells also lose their proliferative capacity in vivo and in vitro [29]. A ip side to these studies concerns whether, in adult cells, miRNAs can stimulate proliferation that leads to a cancerous state. Indeed, more than half of the known human miRNA genes are located near chromosomal breakpoints associated with cancer, and in some documented cases the miRNA genes are amplied, leading to overexpression [30]. For one miRNA cluster, overexpression promotes tumor progression in an animal lymphoma model [31]. Thus, the proliferative potential inherent in miRNAs is a causative agent for cancer.

Biological regulation by miRNAs: single-gene approaches


Another method to determine the functions of miRNAs is to inactivate each miRNA individually (Table 1). Due to the small size of each gene, mutagenesis has been technically challenging, but recent progress has been forthcoming. As one would expect, specic miRNA genes have been found to be important for cell growth, morphogenesis and apoptosis. The largest Drosophila miRNA gene family, the miR-2 family, is required for the suppression of apoptosis during embryogenesis [32]. This observation was made in an elegant study that used antisense RNAs, rather than traditional mutagenesis, to systematically inactivate miRNAs. Human miR-175p and miR20a appear to be regulators of proliferation; they bind and repress E2F1 mRNA in response to stimulation by c-Myc [33]. This might allow for tight control of the proliferative signal. And an evolutionarily conserved function for miR1 in muscle proliferation and morphogenesis was recently described. Muscle-specic expression of miR-1 is activated by the pro-myogenic transcription factor Mef2 in both Drosophila and mouse [34,35]. Murine miR-1 suppresses the proliferation of ventricular cardiomyocytes by downregulating the Hand1 transcription factor [34]. By contrast, loss of Drosophila miR-1 results in abnormal growth and morphogenesis of muscles [35]. The paradigm for this single-gene approach has been C. elegans, in which the rst miRNAs, lin-4 and let-7, were discovered. The miRNA let-7 is expressed late in the nematode life cycle, and it promotes the larva L4-toadult transition in diverse tissues. Several targets of let-7 have been identied in four of these tissues, and all were found to be transcription factors, consistent with the idea that let-7 regulates the timing of differentiation [36]. On the basis of sequence similarity, several other miRNAs are highly related to let-7. Three of these miRNAs function together to control the timing of the L2-to-L3 transition [37,38]. Consistent with these observations, mutants in the C. elegans Dicer gene exhibit similar

Table 1 Summary of selected miRNAs and their biological activities miRNA let-7 miR-48, miR-241, miR-84 lsy-6, miR-273 miR-84 miR-61 miR-2, miR-6, miR-11, miR-308 miR-1 miR-31 miR-7 miR-1 miR-196a miR-17-5p, miR-20 Species Nematode Nematode Nematode Nematode Nematode Fruit y Fruit y Fruit y Fruit y Mouse Mouse Human Target genes daf-12, pha-4, hbl-1 hbl-1 cog-1, die-1 let-60 vav-1 hid, grim, rpr, skl Unknown Unknown Yan Hand1 Hoxb8 E2F1 Function L4adult transition L2L3 transition ASE fate decision Secondary vulva cell fate Secondary vulva cell fate Apoptosis Muscle growth Embryo segmentation Photoreceptor fate Myocyte proliferation Unknown Proliferation References [36] [37,38] [42] [45] [46] [32] [35] [32] [44] [34] [41] [33]

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206 Chromosomes and expression mechanisms

developmental timing defects [39,40]. Thus, miRNAs play an important role in controlling the timing of developmental decisions. A few miRNAs have been found to regulate cell differentiation. Axial patterning of the embryo is aided by miR31 in Drosophila and by miR-196a in the mouse [32,41]. Although miR-31 inuences segmentation by affecting unidentied downstream genes, a target of miR-196a was identied to be Hoxb8, which is consequently restricted from the posterior of the mouse embryo [41]. A more detailed view of the roles played by miRNAs in cell fate determination has come from the study of neuronal development. In C. elegans, two gustatory neurons adopt either an ASEL or ASER fate from a bi-potential ASE precursor. The fate decision is controlled by two miRNAs, lsy-1 and miR-273, which mutually repress each others expression [42]. They do so by inhibiting transcription factors that activate lsy-1 and miR-273 transcription (Figure 1a). This four-component feedback loop is double-negative in character, and it forms a bistable system. Bistable systems exist almost exclusively in one of two possible states that are stabilized by feedback loops [43]. In the case of the ASE decision, the two states are the ASEL and ASER fates. The bistable nature of the feedback mechanism ensures that the adoption of one state is maintained or stabilized. For example, if a weak signal to differentiate is received by an ASE cell, the bistable switch could amplify the signal into a strong uniform response. Thus, it would ensure that signal variation between different cells has less impact on their ability to uniformly respond. Likewise, if a signal to differentiate is transient, the bistable switch could translate the transient signal into a long-lasting response. The relationship between bistable feedback loops and miRNAs is more pervasive than this one example. Expression of the Drosophila miRNA miR-7 is turned on in cells as they begin to differentiate into photoreceptors [44]. This is dependent on EGF receptor (EGFR)-signaling, which triggers ERK (extracellular signal-regulated kinase)-mediated phosphorylation and degradation of the transcription factor Yan. In non-stimulated cells, stabilized Yan represses miR-7 transcription. In turn, miR-7 miRNA represses Yan protein expression in photoreceptors, directly, by binding to complementary sequences within its mRNA 30 UTR. This reciprocal negative feedback between Yan and miR-7 ensures mutually exclusive expression, with Yan in progenitor cells and miR-7 in photoreceptor cells (Figure 1b). Expression is switched when EGFR-signaling transiently triggers Yan degradation. The long-term depletion of Yan from differentiating cells is crucial because it inhibits transcription of multiple cell-specic genes. This mechanism involving miR-7 explains how signal transduction activity can robustly generate a stable change in gene expression patterns in the Drosophila eye.
Current Opinion in Genetics & Development 2006, 16:203208

Figure 1

Cell fate determination and the roles of miRNAs. (a) ASE cell fate determination in the C. elegans nervous system. Two ASE cells undergo fate decisions; the cell on the left side of the animal becomes an ASEL, and the cell on the right becomes an ASER. Adoption of the ASEL fate is determined by miRNA lsy-6, whereas the ASER fate is determined by miRNA miR-273. These RNAs mutually inhibit each others expression by repressing the transcription factors cog-1 and die-1. (b) Photoreceptor cell fate determination in the Drosophila eye. In the absence of an EGF receptor signal, the transcription factor Yan represses expression of the miR-7 gene. When the EGF receptor is activated, Yan protein is degraded, making it incapable of repressing miR-7. The synthesized miR-7 miRNA binds to the 30 UTR of yan transcripts, thereby repressing Yan expression. This reciprocal negative feedback loop between miR-7 and Yan exerts itself upon the differentiation state of cells through the action of Yan upon downstream genes. It is also possible that miR-7 regulates other downstream genes outside of the feedback loop. (c) Vulva cell development in C. elegans. The let-23 EGF receptor is activated in the P6.p VPC, which induces its primary cell fate and, secondarily, leads to secretion of the DSL protein. DSL interacts with its cognate Notch receptor, LIN-12, on the surface of the neighboring P5.p and P7.p cells. Signal transduction through the let-23 pathway is attenuated in these cells by the action of miR-84 on LET-60 Ras expression. Activation of LIN-12 leads to expression of miR-61, which represses Vav-1. Given that the Vav-1 protein normally inhibits LIN-12, its downregulation by miR-61 causes, in essence, a positive feedback loop to be engaged. This helps to switch these cells to a secondary cell fate.

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Gene regulation by microRNAs Carthew 207

Other examples of interactions between miRNAs and signal transduction networks are emerging (Figure 1c). Development of the C. elegans vulva requires cellcell interactions to specify vulval precursor cells (VPCs) into primary, secondary and tertiary fates. An inductive signal from the gonad activates the EGFR pathway in a VPC, causing it to differentiate as a primary cell. The EGFR pathway is suppressed by miR-84, which is expressed in early secondary cells and acts directly upon an EGFR pathway component, Ras [45]. Hence, miR-84 acts to attenuate signaling activity that promotes primary cell fate determination. Once determined, the primary cell produces a lateral signal that is received by the Notch receptor on the surface of neighboring VPCs. When activated, Notch is cleaved, and the intracellular fragment localizes to the VPC nucleus and stimulates transcription of downstream genes. One of these genes encodes miRNA miR-61 [46]. In turn, miR-61 binds to the mRNA encoding Vav-1, the C. elegans ortholog of the Vav oncogene product, and downregulates its expression. Because Vav-1 represses Notch, the downregulation of Vav-1 by miR-61 augments Notch signaling activity and promotes the VPC to adopt a secondary fate. Thus, a reciprocal negative feedback loop between a miRNA and protein in this case, Vav plays an important role in a cell fate decision triggered by a cellcell signal; the mechanism bears a striking resemblance in certain respects to the interaction between miR-7 and Yan.

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Conclusions
It is becoming clear that miRNAs play diverse regulatory roles in animal cells. They might use several mechanisms to repress gene expression, although it is still uncertain if these are related to each other. Cellular activities such as proliferation, morphogenesis, apoptosis and differentiation are regulated by miRNAs, and in some cases, upstream and downstream genes have been linked to the miRNAs. Several miRNAs have been found to act in reciprocal negative feedback loops with protein factors to control cell fate decisions that are triggered by signal transduction activity. It remains to be seen how generally miRNAs will be involved in this type of mechanism. But the potential of rapidly evolving miRNA regulation could be important for evolving new regulatory circuits and, ultimately, new patterns within body plans.

Acknowledgements
This work has been supported by the National Institutes of Health.

References and recommended reading


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