MicroRNAs: key participants in gene regulatory networks Commentary Xi-Song Ke, Chang-Mei Liu, De-Pei Liu and Chih-Chuan Liang

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1 516 MicroRNAs: key participants in gene regulatory networks Commentary Xi-Song Ke, Chang-Mei Liu, De-Pei Liu and Chih-Chuan Liang micrornas (mirnas) are a newly identified and surprisingly large class of endogenous tiny regulatory RNAs. They exhibit various expressional patterns and are highly conserved across species. Recently, several regulatory targets of mirnas have been predicted. Functional analysis of the potential targets indicated that mirnas may be involved in a wide range of pivotally biological events. The nature of mirnas and their intersection with small interfering RNAs endow them with many regulatory advantages over proteins and make them a potent and novel means to regulate gene expression at almost all levels. Here we argue that mirnas are key participants in gene regulatory network. Addresses National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, , China Liudp@95777.com /$ see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI /S (03) Abbreviations B-CLL B-cell chronic lymphocytic leukemia CAF CARPEL Factory mirnas micrornas mirnp micro-ribonucleoprotein ncrnas non-coding RNAs PPD PAZ/PIWI domain RdRP RNA-dependent RNA polymerase RISC RNA-induced silencing complex RNAi RNA silencing sirna small interfering RNA UTR untranslated region Introduction Four decades ago, the Central Dogma was formulated and simplified as DNA makes RNA, and RNA makes protein. As a result, RNAs have been looked as simple molecules that merely convert genetic information into protein. However, it has been estimated that although most of the genome is transcribed, almost 97% of the genome does not encode proteins in higher eukaryotes [1]; it is difficult to believe that these vast transcripts have no function. Now, putative non-coding RNAs (ncrnas) have been discovered that function directly as RNA rather than encoding proteins [2]. ncrnas seem to be particularly suited for roles that require highly specific nucleic-acid recognition. The view of RNA has changed from the purely informational medium to a variety of structural, informational and even catalytic molecules in the cell. Many other ncrnas that function as regulators have also been discovered [3], but their number and importance seem marginal. Recently, a remarkably large number of tiny ncrna genes have been identified and named micrornas (mirnas) [4 6,7,8 10,11,12,13]. The first mirnas to be discovered were the heterochronic regulatory RNAs Lin-4 and Let-7 [14,15]. mirnas have been evolutionarily conserved over a wide spread of species and exhibit diversity in expression profiles, suggesting that they occupy a wide variety of regulatory functions and exert profound effects on cell growth and development [4 6]. Recent work showed that mirnas can regulate gene expression at many levels, offering a novel and promising gene regulatory mechanism and strongly supporting the idea that RNA can perform the same regulatory roles as proteins. Understanding this RNA-based regulation will help us to finally understand the complexity of the genome and the gene regulatory network. Identification mirnas are 22 nt RNAs that arise from one arm of longer endogenous hairpin transcripts [16 ]. Hundreds of mirna genes have been identified in many species, mostly in intergenic regions. Several mirnas that correspond to putative genes have also been identified [5,10,13]. Some mirnas have multiple loci in the genome [9,11 ]; occasionally, several mirna genes are arranged in tandem clusters [4,7,12 ]. Despite the success of recent efforts, the fact that many mirnas have been isolated just once suggests the search for new mirnas is far from complete [9,10,11 ]. An analysis of chromosomes 21 and 22 found that ten times more sequences of the genome were transcribed than predicted. The phylogenic conservation of such transcripts indicates that they do have a function [17 ], and also hints that many genes operate below our radar. The identified mirnas probably represent only the tip of the iceberg, and the number of mirnas might turn out to be very large. Evidence has been presented that mature mirnas are cleaved from their precursors by the RNaseIII family (Dicer) [18]. Taking together all identified mirnas, the characteristics of mirnas can be summarized as following: 1. They are single-stranded RNAs of about 22 nt. 2. They are cleaved from one arm of a longer endogenous double-stranded hairpin precursor by Dicer.

2 MicroRNAs: key participants in gene regulatory networks Ke et al They should match precisely the genomic regions that can potentially encode precursor RNAs in the form of double-stranded hairpins. 4. mirnas and their predicted precursor secondary structures are phylogenetically conserved. 5. mirnas and their potential precursors can be confirmed by northern blots. 6. When Dicer is mutated or knocked-out, mirna precursors accumulate. Figure 1 5 P 3 OH 5 P Hairpin precursor Biosynthesis Much evidence has supported that Dicer and Argonaute are crucial participants in mirna biosynthesis and function. Dicer and Argonaute animal mutants exhibit reduction in mature mirna accumulation, essential reiteration of cell division and delay of the switching to a later stage developmental programme. In mutant Arabidopsis, disruption of embryo development, delay in flowering time and over-proliferation of floral meristems occurs. These lines of evidence suggest both Dicer and Argonaute are indispensable for the maturation and activity of mirnas [9,19]. Argonaute has been shown to interact with mirnas genetically and biochemically, and the translation initiation factor eif2c can cleave substrates that are homologous to its constituent mirnas [7,19 21,22 ]. Mutations in genes required for mirna biosynthesis lead to genetic developmental defects; these dramatic consequences are derived, at least in part, from the role of generating mirnas [22 ]. 3 OH Dicer 5 P 3 OH Co-factors P 5 OH 3 Pre-miRNA Pre-miRNP Degradation Although the details are still obscure, the outline of the mirna biosynthesis pathway is beginning to emerge. In the earliest experiments, mirnas were generated from their precursors by Dicer in animals or its Arabidopsis homologue CARPEL Factory (CAF) [9,11,18]. As several mirna pairs were cloned from both sides of the same precursors [4,5,7,9], these mirnas could base pair with each other and potentially form duplexes with 2 nt overhangs at both 3 0 ends, a feature of the Dicer products [23]. We propose that mirnas are initially cleaved in duplex form rather than from one arm of their precursors. Secondly, the duplex mirnas (pre-mirnas) are transferred to pre-micro-ribonucleoprotein (pre-mirnp), a multi-protein RNA complex whose constituents include Argonaute and RNA helicase [7,12,19] and that differs from mirnp only in that it contains double-stranded mirna [7 ]. Thirdly, cofactors unwind the pre-mirnas into single strands, thus transforming pre-mirnp into mirnp. Argonaute proteins belong to the PAZ/PIWI domain (PPD) family, and PAZ is also a component of Dicer and thought to mediate Argonaute Dicer interaction [24,25]. Finally, mirnas recognize targets whilst sequestered within mirnp. Hence, mirnps may play an important role in the maturation and delivery of their mirnas to RNA targets [12 ], and different effectors could integrate into and lead mirnas to diverse functional pathways (Figure 1). Diverse effectors Functions mirnp Current Opinion in Chemical Biology Model for the biosynthesis and function pathway of mirnas. mirnas are cleaved by Dicer from the hairpin precursor in the form of duplex, initially with 2 or 3 nt overhangs in the 3 0 ends, and are termed premirnas. Cofactors join the pre-mirnp and unwind the pre-mirnas into single-stranded mirnas, and pre-mirnp is transformed to mirnp. mirnas can recognize regulatory targets while part of the mirnp complex. Different effectors lead mirnas into diverse pathways. The structure of pre-mirnas is consistent with the observation that 22 nt RNA duplexes with 2 or 3 nt overhangs at the 3 0 ends are beneficial for reconstitution of the protein complex and might be required for highaffinity binding of the short RNA duplex to the protein components [23]. Functions Detailed functions Growing evidence suggests that mirnas play crucial roles in eukaryotic gene regulation. The first mirnas genes to be discovered, lin-4 and let-7, are heterochronic switching genes that are essential for the normal temporal control of diverse developmental events in Caenorhabditis elegans. Both lin-4 and let-7 base-pair incompletely to

3 518 Commentary repeated elements in the 3 0 untranslated regions (UTRs) of other heterochronic genes, and regulate the translation directly and negatively by antisense RNA RNA interaction [14,15]. Mutations in lin-4 or let-7 causes temporal transformations in cell fates with omission or reiteration of stage-specific events [14,15]. Studies of lin-4 indicated that the stability, polyadenylation level and polysome association of target mrnas were not affected [26,27]. This regulatory model seems flexible and efficient, for the translational repression can be easily deleted just by releasing the mirnas, rather than destruction and synthesis of new mrnas. The other mirnas were thought to interact with target mrnas by limited complementary and suppressed translation as well [4 6]. Many studies have shown, however, that the acting model of lin-4 and let-7 might be just one type of functional mechanism mediated by mirnas. A large subset of Drosophila mirnas were shown to be precisely complementary to the K box, Brd box and GY box motifs in the 3 0 -UTR, and the two former motifs were previously demonstrated to significantly affect both transcript stability and translational efficiency [28 ]. Given a perfectly complementary target RNA, let-7 could direct target RNA degradation rather than inhibit translation [22 ]. In addition, Arabidopsis mir39 /mir171 was perfectly complementary to internal regions of some mrnas, and resulted in specific cleavage of targets within the region of complementarity [29]. This suggests that only the degree of complementarity between mirnas and their RNA targets determines their functions [22 ]. Predicted functions Predicting potential targets is the major challenge in exploring mirna functions. By identifying sequences with near complementarity, several targets have been predicted. Strikingly, most of the potential or putative targets are transcriptional factors that are crucial in cell growth and development. Both the basic-helix loop helix (bhlh) repressors and Bearded family are predicted targets of many mirnas, and mirnas could be the key to explain the plethoric developmental phenotypes [28 ]. The bhlh repressors belong to a superfamily of DNA-binding transcription factors that regulate numerous biological processes, including cell fate decisions in both invertebrates and vertebrates [30]. Bearded family members are modulators of the Notch signaling pathway in multiple cell fate decisions during adult sensory organ development [31]. Scarecrow-like family are targets of mir39/mir171 and regulate an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root [32]. mirnas in Arabidopsis are almost complementary to many sequences. Some of these potential targets are transcriptional factors, including squamousa-promoter-binding-protein-like proteins and CCAAT-binding-factor-HAP2-like proteins, many of which are involved in developmental patterning or cell differentiation, and the complementary sites were conserved among flowing plants [9,33 ]. Predicted targets for mir165 encoded HD-Zip transcription factors. The potential complementary sites might explain the ectopic expression previously described for mutations in these genes. It seemed that complementarity to mir165 was required for confining these mrna s accumulation to the proper cell types [33 ]. Targeting of developmental transcription factors suggested that many plant mirnas function during cellular differentiation to clear key regulatory transcripts from daughter cell lineages [33 ]. Interestingly, Argonaute1, which was required both for axillary shoot meristem formation, leaf development in Arabidopsis and the biosynthesis and functions of mirnas, was a predicted target of mir168, suggesting a negative feedback mechanism to control expression of Argonaute and activities of mirnas [33 ]. The high percentage of predicted mirna targets acting as developmental regulators and the conservation of target sites suggested that mirnas are involved in a wide range of organism development and behaviour and cell fate decisions [33 ]. Analysis of the genomic location of mirnas indicates that they play important roles in human development and disease. For examples, mir10 was located and conserved in the Hox gene cluster in multiple species. Considering the spatial and temporal colinearity of Hox gene expression and the positional conservation of mir-10, it is conceivable that mir-10 is important for regulating developmental events [13]. mir15 and mir16 are clustered and located within the intron of LEU2, which lies within the deleted minimal region of the B-cell chronic lymphocytic leukemia (B-CLL) tumor suppressor locus, and both genes are deleted or down-regulated in the majority of CLL cases [13,34 ]. It is possible that mirna levels are crucial in maintaining normal B cell differentiation. B-CLL suppressor genes have yet to be identified and mir-15 and mir-16 are strong candidates [13]. Proteins related to fragile X syndrome have been associated with protein complexes that contain mirnas. The possibility that these proteins use an RNA-related mechanism for target recognition suggests that defects in mirna-related machinery may cause human disease [35,36]. In addition, the locus of mir-175 is a candidate region for Waisman syndrome and X-linked mental retardation [12 ]. Such findings present further evidence for the involvement of mirnas in human diseases, and the significance of this growing gene family. SiRNA-related functions In addition to mirnas, there are other 22-nt ncrnas named small interfering RNAs (sirnas) [23]. sirnas are derived from exogenous dsrna molecules including very long hairpins or bimolecular duplexes. Numerous sirnas accumulate from both strands of the dsrna after the action of Dicer [16 ]. sirnas function in the form of

4 MicroRNAs: key participants in gene regulatory networks Ke et al. 519 RNA-induced silencing complex (RISC) to mediate the destruction of homologous RNA targets during RNA silencing (RNAi), which was thought to be the immune system of the genome against the proliferation of invading molecules [37]. It was demonstrated that perfect basepairing between sirnas and target mrnas is required for mrna cleavage [38]. Furthermore, sirnas might modulate gene expression through additional mechanisms including guiding cytosine methylation, histone methylation and epigenetic modifications of homologous DNA sequences [39 41]. sirnas and RNAi-related proteins including Dicer, Argonaute and RNA-dependent RNA polymerase (RdRP) appear to be central components of the chromatin-silencing machinery. sirnas are required for the proper regulation of chromosome architecture during mitosis and meiosis in fission yeast [42,43]. Provost s work suggested an evolutionary conserved role of Dicer in chromosome function [44]. The Argonaute family members are important in post-transcriptional and transcriptional gene silencing and programmed DNA elimination [41]. Importantly, sirnas were able to signal intercellularly and trigger silencing systemically throughout the plants [45]. Except for the different origins of their precursors, mirnas and sirnas cannot be distinguished from their biochemical compositions, biosynthesis and functions. The generation and activities of both require Dicer and Argonaute [18,21,46]. In fact, there are several tantalizing similarities between mirnp and RISC, including similar sizes and both containing RNA helicase and the PPD proteins. It is therefore tempting to speculate that mirnp and RISC are the same RNP with multiple functions [47]. In addition, let-7 and mir39 could naturally enter the RNAi pathway and cleave target mrnas [22,29]. The nearperfect complementarity between plant mirnas and their targets suggests that they act similarly to sirnas and direct target RNA cleavage [33 ]. Interestingly, experiments showed that sirnas can function as mirnas to repress expression of a target mrna with partially complementary binding sites in its 3 0 -UTR [48 ]. Furthermore, sirnas could guide target RNA cleavage in RNAi in the form of single strands such as mirnas [49]. On the other hand, Drosophila Argonaute coimmunoprecipitated with both mirnas and sirnas [35]. All these lines of evidence indicate that the two pathways intersect, and may even be the same. In some sense, mirnp/risc could be considered as a flexible platform into which different regulatory modulators could be incorporated. The core complex would be required for receiving small RNAs from Dicer and guiding them to recognize their homologous targets. Different effectors could join in the core. When nucleases are incorporated, they direct the degradation of targets, and translational repressors lead to translational inhibition. The inclusion of chromatin remodeling factors, on the other hand, mediates transcriptional silencing. Other adaptations might also exist [50]. Deduced functions Considering their genomic location, the employment of tiny RNAs to specific recognition and the connection with sirnas, it is reasonable to deduce the almost infinite potential of mirnas in regulating gene expression at nearly all levels. As some mirnas are located less than 1 kb from either 5 0 start or 3 0 end of putative genes that contain many cis-acting elements, even in introns, exons or the complementary spanned splice sites of known protein coding genes [5,13,33 ], it is imaginable that mirnas have regulatory activities ranging from gene transcription as trans-acting factors, translational initiation, pre-mrnas splicing, RNA transport, mrnas stability or localization as the base-pairing site diversion. But it is not clear how to control these RNAs to function as mirnas or traditional RNAs. Some mirnas come from the same precursors [4,5,7,9], indicating that interaction may take place between mirnas. In addition, there are mirnas that are related in sequence to other ncrnas, such as rrnas, trnas or snrnas [10], arguing strongly for a more general function of mrnas in post-transcriptional regulation. Furthermore, as some mirnas correspond to the sense strands of mrnas [5], or are complementary to potential promoter sequences, and CAF has putative nuclear locational signals, it is conceivable that mirnas might guide the modification of functional or structural chromatin DNA sequences and regulate gene expression at the DNA level, such as replication, transcription or epigenetic control of nuclear genomes [9]. Interestingly, mirnas may also target proteins; a good example was a regulatory RNA CsrB, which competed with another mrna to bind a global regulatory protein CsrA, and antagonized its activity [51]. Advantages as regulators As tiny RNA regulators, mirnas seem to be highly adaptable for participators in gene regulating processes. By their very natures, mirnas can do the same as traditional protein regulators and even provide obvious advantages over the later in many repects. Firstly, Watson Crick base pairing between mirnas and target RNAs or DNAs allows an exquisitely specific recognition, a job that RNA is more suited to than proteins. Secondly, mirnas can be generated more rapidly when required, for a mirna precursor is much smaller than a typical protein-coding gene, and without being delayed by translation and post-translation modification in synthesizing. Rapid expression and the ability to act directly as RNA may facilitate the precise temporal regulation, especially in developmental transitions via mirnas. Thirdly, it is evident that production and degradation of mirnas are both more cost-efficient than a specific RNA-or DNAbinding protein domain. Fourthly, there are an enormous number of different theoretical mirnas, and each has many potential targets with different base-pairing modes. The diverse degree and details of base pairing provide a wealth of possibilities and flexibilities in gene regulation.

5 520 Commentary Fifthly, evolution of a specific complementary mirna could be achieved more simply, just in a single step by a partial duplication of a fragment of the target gene into an appropriate context for expression of new genes [2]. Sixthly, many processes in gene regulation can be obtained simply by steric interference of target DNAs, RNAs or proteins, which could be carried out only via mirnas. Finally, given that they share the same mechanism with sirnas to be amplified by RdRP activity [52,53], mirnas may scale-up greatly both in the number and effect. Taking all this together, we can see that mirnas are more optimal materials than proteins in many ways, and may be the simplest molecules for the desired gene regulation. Key participants in gene regulatory network From the above discussion, we can see that mirnas have various expression patterns including stage-specific and/or tissue-specific and uniform during development and are involved in a broad range of key process in cell division, and cellular and organism physiology; vast numbers of mirnas exist; their predicted targets and genomic locations are diverse and interesting; and, most importantly, they have the potential to interact with RNA, DNA and protein molecules to regulate gene expression in a diverse array of developmental or physiological events at almost all levels, including chromatin remodeling, gene transcription, RNA processing and location, translation efficiency, RNA transport, RNA stability, even proteins activity. It is logically concluded that mirnas are key participants even at the second center in a gene regulatory network (Figure 2). Although the details of mirna-mediated gene regulatory networks are not clear yet, the tiny size and prevalence of mirnas hint that they might collaborate with proteins to control gene expression, and mirna-based mechanisms might be widespread in higher eukaryotes. When processes require simply alteration of steric structure of the target molecules to indirectly control the binding of other regulatory factors or blocking of active sites, even tagging on targets to recruit other regulators, mirnas can carry out such roles alone. In cases requiring more sophisticated functions, a small number of shared proteins could be incorporated into a common protein complex containing mirnas, with the mirnas acting as guiders in specific sequence recognition, and the incorporated proteins performing specific functions. In this sense, mirnas could potentially regulate in trans any biological process involving RNA RNA, RNA DNA or RNA protein interactions. In fact, the relatively short length seems insufficient to induce complex structural signals for stability or intracellular transport. In addition to antisense sequence elements, therefore, it is likely that many different mirnas utilize a common protein complex for intracellular trafficking and protection from degradation [54]. It is possible that mirnas could be Figure 2 Pre-mRNA mrna 5,6 7,8 Protein ,6 7,8 1. Chromatin remodeling 2. Gene methylation 3. Transcription 4. RNA process 5. RNA stability 1 Gene Chromatin Precursor mirna trafficked systemically to distant organs like sirnas and contribute to long-distance regulation in response to environmental signals. Demonstration that mirnas traffic systemically and selectively in plants would significantly enrich the emerging paradigm and lay an RNA signaling superhighway to facilitate communication and coordination of vital information for plant growth, development and response to the environment [55]. The discovery of the phylogenic distribution of mirnas implies the ancient origin of the mirna pathway, which arose early in eukaryotic evolution and might have played roles during the origins and evolution of both plant and animal multi-cellular life [11 ]. However, considering the versatility of mirnas and their advantages over peptides for some mechanisms, it is likely that mirnas aren t simply the remnants of ancient, pre- DNA and pre-protein control systems [56], but play ,5 6, RNA transport 7. RNA location 8. Translation 9. Protein activity 10. mirna stability Current Opinion in Chemical Biology mirnas are key participants even at the second center in gene regulatory networks. mirnas potently interact with RNA, DNA and even proteins and regulate gene expression at nearly all steps. Importantly, by targeting Argonaute proteins, a negative feedback mechanism for the biosynthesis and activity of mirnas may exist. Purple arrows indicate activities of proteins and green arrows indicate activities of mirna.

6 MicroRNAs: key participants in gene regulatory networks Ke et al. 521 key roles in higher eukaryotes even now. Although proteins are the known fundamental effectors of cellular function, the basis of eukaryotic complexity and phenotypic variation might lie primarily in a control architecture that is composed of a highly parallel system of transacting RNAs. These RNAs relay information required for the coordination and modulation of gene expression [57]. mirnas might act as ancient but vigorous participants and a second center of gene expression, and enable the integration and completion of the traditional network. Conclusions and future challenges We should be excited by the exploration of the novel regulatory paradigm of mirnas; it opens a new vista and adds another dimension to the diversity and complexity of gene-regulatory networks. mirnas also offer possible explanations for some puzzles in deeply studied but unclear regulatory pathways and complex genetic phenomena such as imprinting and RNA interference that involve RNA signaling. Furthermore, mirnas might help to partly decipher the mystery that higher organisms have striking complexity in phenotypes yet a relatively limited number of protein coding genes in their genomes [57].The field has advanced at an astonishing rate over the past few years; however, the key roles of mirnas are just beginning to be glimpsed and we are far from fully appreciating the mechanism of mirna-mediated gene regulatory networks. In our opinion, the primary challenges for understanding mirna-mediated networks are to verify the predicted targets of mirnas by genetic or biochemical experiments, elucidate the exact mechanism of mirnas working on one another, and identify the pathways that control the expression and modulate the activities of mirnas to respond environmental or cellular signals. Update Recent work has explored more evidence for mirna function. mir-14 has been shown to function as a cell death suppressor in Drosophila [58]. Loss of mir-14 enhanced Reaper-dependent cell death, whereas overexpression suppressed cell death. Another mirna gene bantam simultaneously stimulated cell proliferation and prevented apoptosis, and a pro-apoptotic gene, hid, as a target for regulation by bantam mirna provided an explanation for bantam s anti-apoptotic activity [59 ]. Conversely, a heterochronic gene, lin-57/hbl-1, which ensures that postembryonic developmental events are appropriately timed in C. elegans, is a probable target of let-7 and lin-4 regulation through its 3 0 UTR [60,61]. Another example of a negative feedback mechanism involved in control of activities of mirnas has been detected. The levels of Dicer RNAs were elevated in mirna-defective mutant plants, a sequence near the middle of Dicer mrna was the predicted target for mir162, and Dicer-derived RNA with the properties of a mir162-guided cleavage product was identified [62 ]. These results indicate that Dicer mrna was subject to negative feedback regulation through the activity of a mirna. Acknowledgements This work is supported by the Chinese High-Technology (863) Programme 2001AA and NSFC/RGC References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. 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