The evolution of animal microRNA function

Ryusuke Niwa and Frank J Slack

MicroRNAs (miRNAs) are a large class of small RNAs that function as negative gene regulators in eukaryotes. They regulate diverse biological processes, and bioinformatics data indicate that each miRNA can control hundreds of gene targets, underscoring the potential inuence of miRNAs on almost every genetic pathway. In addition to the roles in ontogeny, recent evidence has suggested the possibility that miRNAs have huge impacts on animal phylogeny. The dramatically expanding repertoire of miRNAs and their targets appears to be associated with major body-plan innovations as well as the emergence of phenotypic variation in closely related species. Research in the area of miRNA phylogenetic conservation and diversity suggests that miRNAs play important roles in animal evolution, by driving phenotypic variation during development.
Addresses Department of Molecular, Cellular and Developmental Biology, KBT 936, Yale University, PO Box 208103, 266 Whitney Avenue, New Haven, CT 06520, USA Corresponding author: Slack, Frank J (frank.slack@yale.edu)

level of regulation, such as by alternative splicing and non-coding RNAs [1,4]. Over the past few years, a new and surprisingly abundant class of RNA regulatory genes known as microRNAs (miRNAs) has been found to confer a novel layer of genetic regulation in cells [510]. miRNAs comprise a large family of 22-nucleotide single-stranded RNAs that silence gene expression by binding to target mRNAs. miRNAmRNA binding usually involves strong base-pairing between the 50 end of a miRNA and its target complementary sequence in the 30 -untranslated region (30 UTR) of an mRNA, while additional base pairings can also contribute to the binding. miRNA binding appears to result in translational repression and, in some cases, degradation of cognate mRNAs, causing partial or full silencing of the respective proteincoding genes. We now know that hundreds of distinct miRNA genes control a range of physiological processes in almost all eukaryotes, including development, growth, differentiation and metabolism [510]. miRNAs are currently estimated to comprise 15% of animal genes, making them one of the most abundant classes of gene regulators [5]. Thus, this discovery introduces a new set of evolutionary mechanisms that has the potential to have profoundly inuenced phenotypic complexity and diversity during animal phylogeny. In this brief review, we summarize recent advance of our knowledge about how the conservation and variety of miRNAs and miRNA targets have evolved, and the potential roles of miRNAs during animal evolution.

Current Opinion in Genetics & Development 2007, 17:145150 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by Tom Misteli and Abby Demburg Available online 20th February 2007 0959-437X/$ see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2007.02.004

Evolution of miRNA families and their expression Introduction

A fundamental principle in molecular biology, the socalled central dogma, is that genes are generally proteincoding and genetic output is almost entirely transacted by proteins. With this view in mind, biologists have worked on the premise that evolution should be characterized by changes in genetic complexity that generate functionally novel proteins. However, an implication from studies in the past two decades is paradoxical: even though morphologically complex animals such as vertebrates possess tissues and organs that are lacking in phylogenetically basal animals, both lower and higher organisms encode a majority of the protein-coding gene families, such as transcription factors and signal transduction molecules [1,2,3]. Thus, the taxonomic distributions of proteincoding genes do not correspond to the dramatic increase in morphological complexity during animal evolution. Therefore, an idea arose that gene expression in the complex metazoan genomes required an additional
www.sciencedirect.com

let-7: a founder member of evolutionally conserved miRNAs

The rst example of an evolutionally conserved miRNA is let-7, which was originally identied as an essential regulator of developmental timing in the nematode Caenorhabditis elegans [11]. Expression of let-7 miRNA is undetectable until the late larval stages in C. elegans, when let-7 directs larval to adult cell fate transitions in many tissues. Soon after the discovery of C. elegans let-7, it was demonstrated that let-7 belongs to a larger gene family that has been amazingly conserved across almost all groups of bilaterally symmetrical animals (bilaterians; Figure 1) [12,13]. Moreover, the temporal expression pattern of let-7 also appears to be highly conserved in all organisms examined. For example, let-7 in the fruit y Drosophila melanogaster is absent from embryonic and larval stages and rst appears at the entry of metamorphosis [12]. In mollusc and a polychaete annelids, let-7 miRNAs are detected in adults but not in larvae. Furthermore, in zebrash and
Current Opinion in Genetics & Development 2007, 17:145150

146 Chromosomes and expression mechanisms

Figure 1

[21]. Lung tumor tissues concurrently display signicantly reduced levels of let-7 and increased levels of RAS protein relative to normal lung tissue, suggesting a mechanism for let-7mediated RAS regulation during lung oncogenesis [22]. Curiously, let-7 RNA is not found in more basal metazoans, including nonbilaterians such as ctenophores, cnidarians and poriferans (sponges), and lower bilaterians such as acoels (Figure 1) [12,13]. This implies that acquisition of the let-7 gene was an essential step of evolution from lower metazoan to a higher bilaterian. It is noteworthy that these lower metazoans do not bear true organs, such as gonads [13]. This observation supports an evolutionary role for let-7 in higher bilaterians to repress hundreds of target genes that are required for juvenile ontology and to subsequently play a role in activating the terminal differentiation of organs, tissues and specic cell types of adults. Further elucidation of let-7 targets throughout the coelmate groups will be crucial for understanding the role of let-7 during animal evolution.

The expansion of the metazoan miRNA repertoire

In addition to let-7, some of the other identied miRNAs are highly conserved during animal evolution in terms of both primary sequences and their function [6,7,9]. For example, mir-1 exhibits muscle-specic expression from C. elegans to human, indicating that it operates during muscle development throughout the animal kingdom [23,24]. Consistent with this proposed function, mir-1 is essential for maintaining muscle ber integrity during Drosophila development [25]. Recent studies using computational approaches have also revealed several episodes of miRNA innovation that correspond to major introductions of developmental complexity during evolution. A large number of miRNA families, including let-7 and mir-1, are acquired at a branch from the basal metazoans to a common ancestor of ecdysozoans, lophotrochozoans and deuterostomes (Figure 1) [3,26]. In addition, major miRNA innovations are observed at the branches leading to the vertebrates and to the placental mammals [26]. Consistent with this, many vertebrate-specic miRNAs are expressed in vertebrate-specic organs, including miR-126 in the blood vessels and miR-206 in the somites [27,28]. Furthermore, multiple unique miRNAs have emerged only in primates as compared with other mammals such as rodents and dogs (Figure 1). Among the 800 total primate miRNAs, at least 336 are primate-specic miRNAs and are not found in other mammals [29] (see also Update). These observations support the hypothesis that lying behind the origin of complexity of higher organisms, such as vertebrates, is the dramatic expansion of the noncoding RNA inventory, including the absolute number of miRwww.sciencedirect.com

Acquisitions of miRNAs in metazoan phylogeny. An abbreviated phylogeny is modified from earlier studies [13,26,40]. Green lines represent animal phyla that do not have let-7 miRNAs [13]. Major innovations of miRNA repertoires are represented by magenta arrowheads, which are based on previous data [26,29]. A class of approximately 20 miRNAs, including let-7, is common to all bilaterians except acoels (a). The next innovation (b) is the addition of about 56 new families of miRNAs at the branch leading to the vertebrates. A third miRNA innovation (c) occurred at the branch leading to the placental (eutherian) mammals, at which time about 40 new miRNA families were acquired. Furthermore, a large number of primate-specific miRNAs are also identified (d). Each of the nematode and arthropod lineages might also have evolved unique miRNA families.

mouse, let-7 miRNAs are undetectable in embryos until a time when adult body plans are conspicuous [12,14,15]. Not only is the let-7 miRNA sequence and temporal regulation maintained across animal phyla but several of the genes controlled by let-7 are also evolutionally conserved [16,17]. Moreover, putative binding sites for let-7 have been identied in the 30 UTRs of the let-7 gene target homologs across animal phyla. For example, the murine and chick orthologs of C. elegans lin-41 are also controlled by let-7-mediated downregulation [14,1820]. Another prominent example of an evolutionarily conserved target of the let-7 family is the RAS family of genes, which are well-known players in cancer
Current Opinion in Genetics & Development 2007, 17:145150

The evolution of animal microRNA function Niwa and Slack 147

NAs, rather than an increase in the protein-encoding inventory [1,4]. Moreover, a comparative genomic study has argued that both ies and vertebrates have increased their respective number of cell types over geologic time in a manner strikingly similar to the acquisition of their respective number of miRNAs [3]. The increasing complexity of gene networks established over the course of animal phylogeny is probably due to miRNAs that regulate a growing number of gene targets, thus driving animal evolution.

miRNA sequence conservation does not imply conservation of expression

Most miRNAs identied to date are expressed in a tissuespecic manner [10]. Based on the high degree of miRNA sequence conservation, it has been assumed that there is also a high degree of expression conservation across animal phyla. However, a recent study has warned against this simplistic assumption [30]. Comparison of the expression of 100 miRNAs in sh, chicken and mouse has revealed that the timing and location of miRNA expression is not strictly conserved. Even between two closely related species, medaka and zebrash, several conserved miRNAs, such as miR-145, miR-205 and miR-454a, exhibit clear spatial expression differences [30]. It is thus attractive to speculate that variations in the expression of conserved miRNAs also play a role in shaping the developmental and physiological differences produced during animal evolution.

targets in these two categories are distinguished by their general tissue-specic expression patterns. Genes with non-conserved target sites tend to be expressed in tissues lacking the corresponding miRNA. By contrast, conserved sites are found preferentially in genes that are coexpressed with their miRNAs [33]. It is proposed [10] that the interaction between miRNAs and their conserved target sites is essential for cell-fate switches and is positively selected for during evolution. By contrast, nonconserved miRNA target sequences may have neutral tness as long as the miRNAs that could bind to them are not expressed in the same tissue [10]. Taken together, the concepts of conserved targeting, non-conserved targeting and targeting avoidance are consistent with the idea that miRNAs inuence the evolution of most mRNAs.
miRNAs and phenotypic variations

Signicant phenotypic variations may emerge by evolving miRNAmRNA 30 UTR interactions. In some instances, polymorphisms that affect miRNAmRNA binding can lead to disease states, whereas in other cases this results in non-deleterious phenotypic changes. Tourettes syndrome is a genetic neuropsychiatric disorder. In some pedigrees, this disease is caused by a frameshift loss-of-function mutation in an open reading frame of SLITRK1 (Slit- and Trk-like 1), which encodes a single-pass transmembrane protein [36]. Patients in other pedigrees have a G-to-A single-base change that maps to the 30 UTR of the SLITRK1 gene. This change replaces a G:U wobble base pair with an A:U WatsonCrick paring in a miR-189 binding site (Figure 2). This mutation stabilizes the interaction between the miR-189 and its binding site and, as a consequence, levels of SLITRK1 translation are reduced compared with those in normal individuals [36]. Another interesting example of a miRNA involved in phenotypic variation is the case of Texel sheep. Texel sheep are renowned for their exceptionally welldeveloped muscles. A locus with a major effect on the muscle mass has been mapped to an interval encompassing the myostatin gene GDF8 [37]. Loss of function in GDF8 causes strong musculature in mice, cattle and humans [38]. Although no polymorphic differences have been found in the protein-coding regions of GDF8 between Texel and control sheep, the Texel GDF8 gene carries a G-to-A substitution in the 30 UTR. This mutation switches the GDF8 mRNA from an antitarget into a target of miR-1 and miR-206, which are highly expressed in muscle (Figure 2), causing decreased GDF8 levels which, in turn, leads to the musculature phenotype [37]. These studies clearly demonstrate that changes in miRNA-based regulation can be expected to make important contributions to phenotypic variation, such as disease susceptibility in man, and to microevolution
Current Opinion in Genetics & Development 2007, 17:145150

Evolution of miRNA target sites in 30 UTR sequences

miRNAs have a signicant impact on the evolution of 30 UTRs

Given that miRNA regulatory systems have a signicant inuence on gene expression, many genes are probably under evolutionary pressure to maintain or avoid miRNA complementary sites in their 30 UTRs [10,31]. For example, genes whose expression patterns exhibit a high degree of overlap with a particular miRNA tend to lack target sites for that miRNA. In addition, broadly and highly expressed housekeeping genes, such as ribosomal-associated genes, seem to avoid miRNA targeting by exclusion of target sites and by maintenance of relatively short 30 UTRs [32]. These data support a concept of antitargets. These are genes expressed at high levels in tissues where miRNAs are expressed, but which seem to avoid having sequences to which those miRNAs could bind [32,33]. Target sequences of miRNAs fall into two categories: conserved and non-conserved. Computer analyses have shown that only one-tenth of these predicted target sites are conserved across species [34,35]. This does not mean that the non-conserved sites are not functional; reporter gene analysis has shown comparable levels of repression mediated by conserved or non-conserved sites [33]. However, large-scale microarray analysis has revealed that
www.sciencedirect.com

148 Chromosomes and expression mechanisms

Figure 2

Two examples of miRNA target polymorphisms and the resulting phenotypic variations: Tourettes syndrome (upper) [36] and Texel sheep (lower) [37]. Each arrowhead indicates a site showing nucleotide polymorphism between normal and mutant. Boxes represent 6-bp seed pairing regions, in which a perfect match between miRNAs and their target is crucial for the miRNAmRNA interaction in many cases [34]. In both cases of Tourettes syndrome and Texel, the G-to-A substitutions make stronger interactions between the miRNAs and their targets, leading to increased inhibition of protein synthesis from the targets and causing the relevant phenotypic variations.

among closely related species. Researchers are currently constructing a new database named Patrocles (http:// www.patrocles.org), which is a compilation of polymorphisms that might affect the interaction between miRNAs and their targets in different organisms [37].

new insight into the underlying mechanisms of animal phylogeny.

Update
Recent work has revealed a large number of new miRNAs in human and chimpanzee brains [41]. This work also showed several interesting proles for the newly identied miRNAs. Even though some miRNAs are not conserved beyond primates, a subset of these species-specic miRNAs is expanded in one species through duplication events. Both this study and the previous one [29] support the idea that a pool of such miRNAs contributes to the diversity of developmental and cellular processes and thus promotes the innovation of new miRNA-containing regulatory pathways during evolution.

Conclusions
Although the importance of miRNAs in animal ontogeny has been rapidly elucidated, much less is known about the phylogenetic aspects of miRNA functions. However, studies in this eld to date support the hypothesis that dramatic expansions of the miRNA repertoire appear to be associated with major body-plan innovations as well as the emergence of phenotypic variation in closely related species during animal evolution. Moreover, it is even possible that we have underestimated the impact of the miRNA regulatory system to animal evolution, because new miRNAs and their targets are discovered every day. Research in this area will undoubtedly provide
Current Opinion in Genetics & Development 2007, 17:145150

Acknowledgements
We thank Atsuo Nishino, Mona Nolde and Sarah Roush for critical reading of the manuscript. RN is supported by a fellowship from the International Human Frontier Science Program Research Organization. FJS is supported by a grant from the National Science Foundation. www.sciencedirect.com

The evolution of animal microRNA function Niwa and Slack 149

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. Mattick JS: RNA regulation: a new genetics? Nat Rev Genet  2004, 5:316-323. In this review article, the author presents a hypothesis that noncoding RNAs, including miRNAs, are the main output of the genomes of complex organisms. The author also argues that this hypothesis might be used to understand the true basis of the evolution of organisms and the basis of diversity of individuals and species. For example, Figure 1 in the review nicely shows that the ratio of noncoding to protein-coding DNA increases as a function of developmental complexity. 2. Technau U, Rudd S, Maxwell P, Gordon PM, Saina M, Grasso LC, Hayward DC, Sensen CW, Saint R, Holstein TW et al.: Maintenance of ancestral complexity and nonmetazoan genes in two basal cnidarians. Trends Genet 2005, 21:633-639.

family of miRNAs in mammals begins in the later stages of development. However, although the temporal expression of mature let-7 in C. elegans is mainly regulated at the transcriptional level [39], the authors show that the temporal expression of the mammalian let-7 is not coupled to the transcription of the pri-miRNAs, but is controlled by post-transcriptional regulation. These data indicate that the regulatory step to produce mature miRNAs could be different across animal phyla, even though miRNAs belong to the same conserved family and their spatial and/or temporal expression patterns are supercially the same. 16. Grosshans H, Johnson T, Reinert KL, Gerstein M, Slack FJ: The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans. Dev Cell 2005, 8:321-330. 17. Lall S, Grun D, Krek A, Chen K, Wang YL, Dewey CN, Sood P, Colombo T, Bray N, Macmenamin P et al.: A genome-wide map of conserved microRNA targets in C. elegans. Curr Biol 2006, 16:460-471. 18. Slack FJ, Basson M, Liu Z, Ambros V, Horvitz HR, Ruvkun G: The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol Cell 2000, 5:659-669. 19. Lancman JJ, Caruccio NC, Harfe BD, Pasquinelli AE, Schageman JJ, Pertsemlidis A, Fallon JF: Analysis of the regulation of lin-41 during chick and mouse limb development. Dev Dyn 2005, 234:948-960. 20. Kanamoto T, Terada K, Yoshikawa H, Furukawa T: Cloning and regulation of the vertebrate homologue of lin-41 that functions as a heterochronic gene in Caenorhabditis elegans. Dev Dyn 2006, 235:1142-1149. 21. Esquela-Kerscher A, Slack FJ: Oncomirs microRNAs with a role in cancer. Nat Rev Cancer 2006, 6:259-269. 22. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, Slack FJ: RAS is regulated by the let-7 microRNA family. Cell 2005, 120:635-647. 23. Brennecke J, Stark A, Cohen SM: Not miR-ly muscular: microRNAs and muscle development. Genes Dev 2005, 19:2261-2264. 24. Nguyen HT, Frasch M: MicroRNAs in muscle differentiation: lessons from Drosophila and beyond. Curr Opin Genet Dev 2006, 16:533-539. 25. Sokol NS, Ambros V: Mesodermally expressed Drosophila microRNA-1 is regulated by Twist and is required in muscles during larval growth. Genes Dev 2005, 19:2343-2354. 26. Hertel J, Lindemeyer M, Missal K, Fried C, Tanzer A, Flamm C,  Hofacker IL, Stadler PF: The expansion of the metazoan microRNA repertoire. BMC Genomics 2006, 7:25. This study is the rst intensive description of how miRNAs were acquired during the metazoan evolution. The authors perform a comprehensive study of the phylogenetic distribution and evolutionary histories of the currently known miRNAs and their homologs. They identied three episodes of miRNA acquisitions that correspond to major developmental innovations during evolution: at the branches leading to bilaterians, to vertebrates and to placental mammals. In addition, they observe an interesting expansion of miRNAs in early vertebrates and in an ancestral teleost owing to genome duplications. The reader should read this paper in conjunction with that of Sempere et al. [3]. 27. Wienholds E, Kloosterman WP, Miska E, Alvarez-Saavedra E, Berezikov E, de Bruijn E, Horvitz HR, Kauppinen S, Plasterk RH: MicroRNA expression in zebrash embryonic development. Science 2005, 309:310-311. 28. Kloosterman WP, Wienholds E, de Bruijn E, Kauppinen S, Plasterk RH: In situ detection of miRNAs in animal embryos using LNA-modied oligonucleotide probes. Nat Methods 2006, 3:27-29. 29. Bentwich I, Avniel A, Karov Y, Aharonov R, Gilad S, Barad O,  Barzilai A, Einat P, Einav U, Meiri E et al.: Identication of hundreds of conserved and nonconserved human microRNAs. Nat Genet 2005, 37:766-770. The authors have developed new miRNA discovery tools that combine bioinformatic predictions with microarray analysis and sequence-directed cloning. They successfully cloned a large number of new human miRNAs that were missed by ordinary methods. They estimated that the Current Opinion in Genetics & Development 2007, 17:145150

3. 

Sempere LF, Cole CN, McPeek MA, Peterson KJ: The phylogenetic distribution of metazoan microRNAs: insights into evolutionary complexity and constraint. J Exp Zoolog B Mol Dev Evol 2006, 306:575-588. Based on data from computational analyses and northern blots, the authors hypothesize potential roles for miRNAs in shaping the evolution of morphological complexity across animals. They argue that evolutionary lineages should acquire new miRNAs over time and that there should be an increase in the diversity of miRNAs in lineages as the phenotypic complexity of lineages increases. By joining recent molecular biological data with classical knowledge of morphology and evolutionary biology, the authors present a wide-ranging and thought-provoking discussion (see also [26]). Mattick JS, Makunin IV: Non-coding RNA. Hum Mol Genet 2006, 15 Spec No 1:R17R29. Lim LP, Glasner ME, Yekta S, Burge CB, Bartel DP: Vertebrate microRNA genes. Science 2003, 299:1540. Ambros V: The functions of animal microRNAs. Nature 2004, 431:350-355. Bartel DP, Chen CZ: Micromanagers of gene expression: the potentially widespread inuence of metazoan microRNAs. Nat Rev Genet 2004, 5:396-400. Kidner CA, Martienssen RA: The developmental role of microRNA in plants. Curr Opin Plant Biol 2005, 8:38-44. Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R: Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev 2006, 20:515-524.

4. 5. 6. 7.

8. 9.

10. Plasterk RH: MicroRNAs in animal development. Cell 2006, 124:877-881. 11. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G: The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 2000, 403:901-906. 12. Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, Hayward DC, Ball EE, Degnan B, Muller P et al.: Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 2000, 408:86-89. 13. Pasquinelli AE, McCoy A, Jimenez E, Salo E, Ruvkun G, Martindale MQ, Baguna J: Expression of the 22 nucleotide let-7 heterochronic RNA throughout the Metazoa: a role in life history evolution? Evol Dev 2003, 5:372-378. 14. Schulman BR, Esquela-Kerscher A, Slack FJ: Reciprocal expression of lin-41 and the microRNAs let-7 and mir-125 during mouse embryogenesis. Dev Dyn 2005, 234:1046-1054. 15. Thomson JM, Newman M, Parker JS, Morin-Kensicki EM,  Wright T, Hammond SM: Extensive post-transcriptional regulation of microRNAs and its implications for cancer. Genes Dev 2006, 20:2202-2207. The full implications of this study were not included in our review, but the authors show interesting data for the expression of mammalian miRNAs including let-7. Similar to C. elegans let-7, the expression of the let-7 www.sciencedirect.com

150 Chromosomes and expression mechanisms

total number of human miRNAs is at least 800, larger than initially believed. Furthermore, several of the newly identied miRNAs are specic to primates and are not conserved across mammals. These data support the idea that miRNAs have a key role in the evolution of complexity of higher mammals, such as humans. 30. Ason B, Darnell DK, Wittbrodt B, Berezikov E, Kloosterman WP,  Wittbrodt J, Antin PB, Plasterk RH: Differences in vertebrate microRNA expression. Proc Natl Acad Sci USA 2006, 103:14385-14389. By comparing the expression of many evolutionally conserved miRNAs in medaka and chicken with existing data for zebrash and mouse, the authors concluded that the spatial and temporal expression patterns of these conserved miRNAs are not strictly conserved. They also argue that differences in expression of some of miRNAs are associated with changes in miRNA copy number and/or differences in genomic context between species. 31. Massirer KB, Pasquinelli AE: The evolving role of microRNAs in animal gene expression. Bioessays 2006, 28:449-452. 32. Stark A, Brennecke J, Bushati N, Russell RB, Cohen SM:  Animal MicroRNAs confer robustness to gene expression and have a signicant impact on 30 UTR evolution. Cell 2005, 123:1133-1146. The authors extensively analyzed expression of predicted miRNAs and their cognate targets in several Drosophila species. They report that a large set of genes involved in basic cellular processes avoid miRNA regulation by utilizing short 30 UTRs that are specically depleted of miRNA binding sites. In addition to the study by Farh et al. [33], this report nds evidence for antitargets, which are genes expressed at high levels in tissues where miRNAs are expressed, and which seem to lack miRNA binding sites. This mutually exclusive expression argues that miRNAs confer robustness to tissue-specic gene expression by blocking potentially large sets of mRNAs whose presence would be harmful to the tissue. 33. Farh KK, Grimson A, Jan C, Lewis BP, Johnston WK, Lim LP,  Burge CB, Bartel DP: The widespread impact of mammalian MicroRNAs on mRNA repression and evolution. Science 2005, 310:1817-1821. These authors examine global tissue expression proles of miRNAs and their targets in mice. Strikingly similar to the study by Stark et al. [32], they found that mRNAs co-expressed with miRNAs have evolved to avoid targeting by these miRNAs. A unique point demonstrated by this study is the functional signicance of the difference between conserved and nonconserved miRNA target sites. Both this study and that by Stark et al. [32] essentially come to the same conclusion that miRNAs have had a widespread inuence on spatial and temporal gene expression patterns in organisms. 34. Lewis BP, Burge CB, Bartel DP: Conserved seed pairing, often anked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005, 120:15-20. 35. Krek A, Grun D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, MacMenamin P, da Piedade I, Gunsalus KC, Stoffel M et al.: Combinatorial microRNA target predictions. Nat Genet 2005, 37:495-500.

36. Abelson JF, Kwan KY, ORoak BJ, Baek DY, Stillman AA,  Morgan TM, Mathews CA, Pauls DL, Rasin MR, Gunel M et al.: Sequence variants in SLITRK1 are associated with Tourettes syndrome. Science 2005, 310:317-320. This study shows genetic evidence that Tourettes syndrome, a genetically inuenced developmental neuropsychiatric disorder, is associated with the loss of function of the Slit- and Trk-like 1 (SLITRK1) gene. In addition to a frameshift mutation causing a truncated SLITRK1 protein in some patients, the authors have found independent occurrences of the identical variant binding site for miR-189 in another unrelated patient. The variation inhibits the translation of the SLITRK1 protein more potently than normal, potentially resulting in the cause of the disorder. This study provides us with the rst example that changes in miRNA-based regulation could make crucial contributions to human diseases. 37. Clop A, Marcq F, Takeda H, Pirottin D, Tordoir X, Bibe B,  Bouix J, Caiment F, Elsen JM, Eychenne F et al.: A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nat Genet 2006, 38:813-818. The authors performed quantitative trait loci mapping to identify the gene responsible for the hypertrophically muscled sheep strain called Texel. The authors nicely reveal that a GDF8 (myostatin) allele in the Texel sheep is characterized by a nucleotide transition in the 30 UTR that creates a target site for binding of miR-1 and miR-206. Given that these miRNAs are highly expressed in skeletal muscle, the substitution causes a reduction of the GDF8 protein levels and presumably contributes to the muscular hypertrophy of the sheep. Furthermore, the group of authors has started constructing a new SNP database (Patrocles) to evaluate the frequency of polymorphic miRNAtarget interactions. Their analyses, as well as those of Abelson et al. [36], suggest that mutations creating or diminishing miRNA binding sites in targets are important for generating phenotypic variations. 38. Dominique JE, Gerard C: Myostatin regulation of muscle development: molecular basis, natural mutations, physiopathological aspects. Exp Cell Res 2006, 312:2401-2414. 39. Johnson SM, Lin SY, Slack FJ: The time of appearance of the C. elegans let-7 microRNA is transcriptionally controlled utilizing a temporal regulatory element in its promoter. Dev Biol 2003, 259:364-379. 40. Delsuc F, Brinkmann H, Chourrout D, Philippe H: Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 2006, 439:965-968. 41. Berezikov E, Thuemmler F, van Laake LW, Kondova I, Bontrop R,  Cuppen E, Plasterk RH: Diversity of microRNAs in human and chimpanzee brain. Nat Genet 2006, 38:1375-1377. In addition to the previous study by Bentwich et al. [29], this work compares the miRNA content in the brains of primates such as human and chimpanzee by massive sequencing. Among all of miRNAs in this study, 75% of known human miRNAs cloned were conserved in vertebrates and mammals, 14% were conserved in invertebrates, 10% were primate-specic and 1% were human-specic. These data demonstrate that a certain number of the miRNAs are not evolutionally conserved and might have species-specic function(s).

Current Opinion in Genetics & Development 2007, 17:145150

www.sciencedirect.com