Expanding roles for mirnas and sirnas in cell regulation Kenji Nakahara and Richard W Carthew 1

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1 Expanding roles for mirnas and sirnas in cell regulation Kenji Nakahara and Richard W Carthew 1 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 cell proliferation, apoptosis and differentiation. Moreover, the microrna mechanism is an efficient means to regulate production of a diverse range of proteins. As new micrornas and their mrna targets rapidly emerge, it is becoming apparent that RNA-based regulation of mrnas may rival ubiquitination as a mechanism to control protein levels. Addresses Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA 1 r-carthew@northwestern.edu This review comes from a themed issue on Cell regulation Edited by Craig Montell and Peter Devreotes /$ see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI /j.ceb Abbreviations DCL Dicer-like mirna microrna mirnp micro ribonucleoprotein complex RISC RNA-induced silencing complex sirna short interfering RNA Introduction Gene expression influences so many cellular activities that the study of gene regulation has exerted a siren song over many of us who are interested in cell biology. Although the role that proteins play in this process is well understood, it is becoming clear that small RNAs are also important gene regulatory factors. Small RNAs, including micrornas (mirnas) and short interfering RNAs (sirnas), are components of a RNA-based mechanism of gene regulation found in eukaryotes [1]. sirnas are utilized throughout the Eukaryota to inhibit viruses and transposable elements [2,3]. They also play a role in chromosome organization and in silencing the expression of protein-coding genes [4 7]. The mirna branch of RNA-based gene regulation is less widespread; mirnas are found in plants and animals but are apparently absent in fungi such Schizosaccharomyces pombe [5]. Another class of small RNAs, tiny noncoding RNAs (tncrnas), are related in structure to sirnas and mirnas, but their function is unknown [8,9]. sirnas sirnas have a specific size of 22 nucleotides and are produced from double-stranded RNA precursor molecules of varying length and origin [2]. Precursor RNAs are processed by members of the RNase III family of Dicer or Dicer-like (DCL) enzymes [1]; the resulting sirnas are duplex in structure. They are then incorporated into a RNA-induced silencing complex (RISC) composed of numerous cellular proteins [10]. Incorporation is coupled with duplex unwinding to generate two single-stranded sirnas, of which only one remains associated with RISC [11,12]. Either sirna strand can be competent to associate with RISC, although preference is given to strands whose 5 0 ends base-pair more weakly to their complements [13,14 ], and thus some sirnas can exhibit strong bias towards retaining one strand versus the other within RISC. The retained RNA strand acts as a guide for RISC to find mrna transcripts with complementary sequence (Figure 1). If such mrna molecules are found, the base-pairing interactions between sirna and mrna lead to transcript cleavage and degradation [10,15]. sirnas may also guide factors that methylate histones and DNA, resulting in transcriptional silencing [6,7]. mirnas and their biogenesis Although chemically similar to sirnas, mirnas are formed by Dicer cleavage of hairpin-loop RNA precursors rather than long double-stranded RNAs [1]. These precursors are transcribed from genes within the genome. The first two mirna genes were identified by forward genetics [16,17], and other mirna genes have been identified either by direct cloning of processed mirnas [9,18 24] or by computational prediction and validation [8,25 27]. The number of mirna genes found in three different animal species roughly corresponds to 0.5 1% of the total number of genes in their genomes. The nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster have perhaps mirna genes, and humans have mirna genes. At least 0.2% of the genes of Arabidopsis thaliana encode mirnas, and this number is likely to be an underestimate as its genome has not been subjected to computational examination. The relative representation of mirna genes (0.2 1%) is comparable to families of other gene regulators, such as DNA-binding transcription factors. Most mirna genes are conserved between related species, and 30% of mirna genes are highly conserved, with orthologs found in vertebrate and invertebrate genomes. This suggests that a significant fraction have evolutionarily conserved biological functions (Table 1).

2 128 Cell regulation Figure 1 Heterochromatin HMT RISC sirna Most mirna genes are distinct from other types of genes, and are usually located some distance from them. They are sometimes organized into genomic clusters, with individual members of a cluster exhibiting coordinate expression. This indicates a possible operon-like organization for some mirna genes. Interestingly, a small fraction (10%) of mirnas are located within introns of annotated genes in the same orientation as the predicted mrnas [8,24], which suggests that some mirnas originate from excised introns of pre-mrna transcripts. mrna turnover Translation mirnp mirnp mirna Current Opinion in Cell Biology Small RNAs act upstream of several effectors. sirnas regulate histone methyltransferases (HMTs) in nuclear complexes to promote heterochromatin formation, and they regulate the RISC to degrade specific mrna transcripts. mirnas regulate mirnp complexes that degrade specific mrnas or repress their translation. How different cellular processes are regulated by the same class of small RNA is not currently understood. Biogenesis of mirnas from primary transcripts involves several steps (Figure 2). Primary mirna transcripts are processed in the nucleus by an RNase III enzyme termed Drosha [28 ]. The resulting hairpin-loop RNA ranges in size from nucleotides and folds into a structure with multiple bulges and mismatches [9,21,24]. This pre-mirna is then exported from nucleus to cytoplasm in a RanGTP-dependent manner via a direct association with Exportin-5 [29]. In the cytoplasm, premirna is processed into a nucleotide mirna by Dicer [28 ]. The fully mature mirna accumulates as a single-stranded species from one arm of each mirna hairpin precursor. mirna incorporates into a ribonucleoprotein complex (mirnp) that is similar, and possibly identical, to RISC [23,30,31]. It is suspected that a short-lived duplex intermediate is generated by Dicer, and is unwound as it is assembled into mirnp, preferentially retaining one strand rather than the other according to the same 5 0 -stability rules described for sirnas [13,14 ]. Table 1 Summary of selected Drosophila mirnas and their predicted gene targets. mirna Orthologs in other species Some predicted Drosophila target genes Bantam Nematode bw, deltex, Dll, hid, kelch, RPLR, salm, Scr let-7 Nematode, human dpld, Lar, tamo mir-1 Nematode, human Ac3, Adk2, did, par-6, Sap47, Sap-r, Tm2, Tsf2 mir-2a Nematode amon, AtpBagr, Ets65A, GLaz, grim, Ptp4E, rpr, Scr, skl, tsl mir-7 Human aop, Calx, disp, E(spl)m3/m4, fax, h, hep, Mkp3, MTA1-like, Tom, Vha55 mir-10 Human Doc, JhI-21, Nmda1, Scr, Sps2, Tom mir-33 Human dock, dpld, sli, Stat92E, svp mir-34 Nematode, human Eip71C, fz2, lmg, SoxN, vvl mir-79 Nematode crol, puc, Ubc-E2H, WASp mir-100 Human Asph, Eaat1, Eh, Gclc, Pc, PTP-ER, Sps2, woc mir-124 Nematode, human AtpBagr, ATPCL, Doc3, dranga, grn, insc, sano, svp, trr mir-125 Nematode(lin-4), human Dscam, E2f, ftz, Scr, SerT, Tom mir-133 Human ATPCL, eif3p66, ex, M(2)21AB mir-184 Human ana, cad, FerHCH, oaf, osp, Pkc53A, Sema1b, trio, twi, Vha16, VhaSFD mir-263a Human abd-a, Glycogenin, GP150, jim, mew, oaf, Oli mir-263b Human Caki, lgs, Prosap, smi35a, tws mir-276b Human Cyp18a1, disp, Ef1Bggr, Fib, huntingtin, hth, PFE mir-285 Human beat-iv, heix, sbb, sca Abbreviated gene names are used. Underlined genes have been detected in more than one independent search. Genes marked in bold have been experimentally verified to be mirna targets.

3 Expanding roles for mirnas and sirnas in cell regulation Nakahara and Carthew 129 Figure 2 pre-mirna Exp5 Nucleus Cytoplasm Dicer GTP Ran Ran Drosha Exp5 mirna gene GDP pri-mirna mirnp mirna activities Like sirnas, mirnas repress gene expression by negatively regulating complementary mrnas. Plant mir- NAs generally trigger target mrna degradation by basepairing with near-perfect complementarity [32 34,35 ]. Conversely, several mirnas from animals repress gene expression primarily by blocking the translation of mrna transcripts into protein [17,36 38,39 ]. They interact with their targets by imperfect base-pairing to mrna sequences within 3 0 untranslated regions. The exact mechanism by which they block translation is unknown, although one mirna has been shown to interfere with a post-initiation step in translation [37]. Despite the differences in how mirnas and sirnas 5 1 Current Opinion in Cell Biology Model of mirna biogenesis. Step 1: Transcription of a mirna gene produces a primary nuclear transcript (pri-mirna). Step 2: The nuclear RNase III enzyme Drosha cleaves pri-mirna to generate a stem-loop pre-mirna. Step 3: The pre-mirna directly associates with the exportin Exp5 and Ran-GTP, and this complex exits the nucleus. Hydrolysis of Ran-GTP to Ran-GDP causes dissociation of the export complex. Step 4: The pre-mirna associates with the cytoplasmic RNase III enzyme Dicer, which cleaves it to form a 22-nucleotide mirna intermediate. Step 5: The mirna intermediate rapidly unwinds as it assembles into a mirnp complex, and one mirna strand is retained in the mirnp. function in animals, they are functionally interchangeable if presented with the appropriate target mrna. A mirna will direct mrna cleavage if the target transcript is perfectly complementary in sequence [30,38]. Conversely, a sirna will block translation if the target transcript has partial complementarity [40,41 ].These observations imply that the extent of base-pairing between small RNA and mrna determines the outcome of silencing. It is unclear whether a single silencing complex is competent both to cleave mrna and to block translation, or whether a mirna (or sirna) associates with two biochemically distinct RISC complexes oneabletocleavemrna,andanotherableto block translation (Figure 1). Biological regulation by mirnas A question of great interest concerns the function of mirnas in metazoans. Although the functions of only a few mirnas are known, evidence suggests that mirnas play diverse but important roles in the development of organisms (Table 1). Mutations in Dicer genes have been isolated in several plant and animal species and, as predicted, these mutants exhibit severely reduced levels of mirnas. Partial loss of dcl1 activity in Arabidopsis results in delayed flower timing, loss of meristem identity, and deregulation of meristem stem cells [22]. Complete loss of dcl1 results in embryonic arrest. dcr-1 mutants in C. elegans exhibit defects in the timing of specific cell fate switches during post-embryonic development [42 44]. Knockout of mouse Dicer1 results in embryos depleted of stem cells [45]. These observations suggest that mirnas regulate stem cells, organ differentiation and developmental timing. They appear to regulate events throughout the life cycle, including embryonic and post-embryonic stages (Figure 3). Consistent with a diverse role for mirnas in development, a variety of mirnas exhibit temporal and tissue-specific expression patterns [8,9,18,19,21,39,46 50]. A few mirna genes have been subject to mutation, and these have provided further insights. Two C. elegans mirna genes, lin-4 and let-7, are required for cell-fate switches at specific times during larval development [16,17,36]. This correlates with their expression, which is upregulated at specific stages of the worm life cycle. The Drosophila mirna gene bantam represses apoptosis and promotes cell proliferation in the developing fly, leading to a normal-sized adult [39 ]. Expression of bantam is temporally and spatially controlled in response to signals that pattern adult tissues, suggesting that bantam regulates cell growth responses to external signals during pattern formation. Another Drosophila mirna gene, mir-14, also antagonizes apoptosis during development [51]. Two Arabidopsis mirna genes have also been genetically analyzed. mir-jaw regulates leaf morphogenesis and the timing of flowering [35 ], and mir-172 overexpression leads to early flowering and defective floral

4 130 Cell regulation Figure 3 dicer-1 /dicer-1 Bearded reporter Background Merge Current Opinion in Cell Biology Repression of gene expression by Drosophila Dicer-1 in the developing compound eye. Eye-antennal imaginal discs were stained with antibody to b-galactosidase to detect expression of a LacZ reporter gene fused to the 3 0 UTR of the Drosophila Bearded gene. This 3 0 UTR contains multiple mirna-binding sites. The reporter gene expression (top right; red in merged image, bottom right) is detected predominantly in the region of the eye undergoing extensive photoreceptor differentiation. Clones of cells mutant for the dicer-1 gene (top left; green in merged image) are scattered amongst unmarked dicer-1 þ tissue. Background staining (bottom left; blue in merged image) highlights all cells in the antennal and eye discs. Strong de-repression of reporter gene expression occurs in dicer-1 mutant cells located in the differentiating region of the eye, which in the merged image appear yellow or white. Mutant cells in other regions of the eye and antenna maintain lower levels of reporter gene expression. development [52]. In mouse, mir-181 is preferentially expressed in the B-cell lineage of the hematopoietic compartment, and ectopic expression of mir-181 in hematopoietic progenitors results in enhanced numbers of B cells in vivo [53]. Considerable effort has been spent in finding the direct targets of mirnas in various species. One approach has been to examine genes with related activities for potential mirna interactions. For example, lin-14, lin-2, lin-41 and hbl-1 are key regulators of developmental timing in C. elegans, and transcripts of these genes are translationally repressed by interaction with lin-4 or let-7 mirnas [16,17,36,54,55]. Likewise, in Drosophila, translation of the apoptosis-promoting gene hid is down-regulated by bantam mirna, possibly accounting for bantam s antiapoptotic function [39 ].InArabidopsis, genes important for meristem growth or identity and organ polarity have been identified as mirna targets. These include the Dicer gene DCL, as well as Apetala2, Phavoluta and Phabulosa, which encode transcription factors [21,32, 33,56]. Interestingly, some Apetala2-like genes are translationally repressed by mir-172, representing the first documented plant mrnas that are not cleaved by cognate mirnas [52]. Other transcription factor genes, homologs of CIN, are repressed by mir-jaw in developing leaf tissue [35 ]. Cleavage of their mrna transcripts correlates with a transition from cell division to differentiation, indicating that is required for normal control of leaf growth. The list of known mirna genes is now in the hundreds, and experimental identification of their targets could prove a monumental task. An alternative approach

5 Expanding roles for mirnas and sirnas in cell regulation Nakahara and Carthew 131 has been to predict target mrnas computationally [32,57,58 ]. This has been undertaken by finding conserved sequences within 3 0 UTRs that are thermodynamically favored to interact with mirnas. One screen of Drosophila genes has discovered clusters of functionally related targets for specific mirnas [57]. Several predicted targets were experimentally validated, demonstrating the predictive power of a computational search. A broader screen of 10,000 Drosophila genes has yielded 700 potential targets of 73 mirnas, corresponding to an average of ten targets per mirna [58 ]. Many single targets contain multiple binding sites for one or more distinct mirnas, suggesting cooperative regulation. What is most striking about this analysis is that certain functional and biochemical classes of genes are highly represented as predicted targets, whereas other classes are virtually absent (Table 1). mirnas are predicted to regulate many factors involved in cell shape control and cell adhesion. Transcription factors, particularly members of the Hox family, are predicted to be targets of several distinct mirnas. Regulation of transcription factors appears to be conserved as they are also highly represented as mirna targets in plants [32]. Components of the ecdysone signal transduction pathway, genes involved in morphogenetic and stress signaling, and genes in the cell death pathway are predicted targets. Perhaps the most striking group of putative target genes is one that comprises synaptic proteins and regulators of neural differentiation and connectivity. It suggests that the nervous system is a major site for mirna regulation. Indeed, Drosophila Fragile X protein, a synapse-localized translational regulator, associates with mirnas in RISC [31,60]. Neurons may be particularly dependent upon mirnabased regulation because protein expression in synapses or growth cones can occur far from the nucleus, making it difficult for neurons to rapidly and coordinately regulate gene expression via transcription. Conclusions It is most likely that gene silencing based on sirnas arose early in eukaryote evolution. This then provided the means for metazoans to evolve another mechanism based on endogenous genes encoding mirnas. Given the many levels of gene regulation that already existed when Metazoa first appeared, why did the mirna-based mechanism arise? In plants, most mirnas appear to degrade target mrnas using a sirna-like mechanism. This implies that a single plant mirna molecule can repress multiple transcripts, and a single interactive event is sufficient to remove a transcript. Thus, mirnas can rapidly and completely clear targets from a cell, allowing for rapid transitions in gene expression. As plant targets are often transcription factors that regulate cell differentiation, this enables cells to respond quickly to developmental decisions. In this respect, mirnas may work analogously to protein-degrading mechanisms that operate during animal cell division and differentiation. In animal cells, mirnas regulate their targets by a cooperative mechanism [40 ]. Thus, it seems plausible that the primary purpose of animal mirnas is to titrate gene expression, potentially modulating target protein levels over a large range, depending on the composition and concentration of mirnas. This would afford cells and tissues a powerful ability to coordinate gene expression. Another potentially important consequence would be the ability of mirnas to fine-tune protein levels of target genes. This might be particularly critical for proteins such as transcription factors and components or regulators of multi-protein complexes, which often function cooperatively themselves. Thus, modest changes in the expression of targeted genes could lead to major changes in output. The challenge for the future will be to place mirna-based regulation into relevant signaling pathways, between upstream signals and downstream effectors. Acknowledgements The authors thank Takashi Hayashi for help with Figure 3, and Erik Sontheimer for helpful comments. The authors are supported by a grant from the National Institutes of Health GM 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. Hannon GJ: RNA interference. Nature 2002, 418: Carthew RW: Gene silencing by double-stranded RNA. 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7 Expanding roles for mirnas and sirnas in cell regulation Nakahara and Carthew Xu P, Vernooy SY, Guo M, Hay BA: The Drosophila microrna Mir-14 suppresses cell death and is required for normal fat metabolism. Curr Biol 2003, 13: Aukerman MJ, Sakai H: Regulation of flowering time and floral organ identity by a microrna and its APETALA2-like target genes. Plant Cell 2003, 15: Chen CZ, Li L, Lodish HF, Bartel DP: MicroRNAs modulate hematopoietic lineage differentiation. Science 2004, 303: Abrahante JE, Daul AL, Li M, Volk ML, Tennessen JM, Miller EA, Rougvie AE: The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by micrornas. Dev Cell 2003, 4: Lin SY, Johnson SM, Abraham M, Vella MC, Pasquinelli A, Gamberi C, Gottlieb E, Slack FJ: The C. elegans hunchback homolog, hbl-1, controls temporal patterning and is a probable microrna target. Dev Cell 2003, 4: Xie Z, Kasschau KD, Carrington JC: Negative feedback regulation of Dicer-Like1 in Arabidopsis by microrna-guided mrna degradation. Curr Biol 2003, 13: Stark A, Brennecke J, Russell RB, Cohen SM: Identification of Drosophila microrna targets. PLoS Biol 2003, 1: Enright A, John B, Gaul U, Tuschl T, Sander S, Marks D: MicroRNA targets in Drosophila. Genome Biol 2003, 5:R1. This paper describes the use of a computational approach to find potential mrnas that are regulated by 73 distinct mirnas in Drosophila. The 700 targets from the screen are highly enriched in certain functional groups including transcription factors, translation and ubiquitination factors, regulators of the cytoskeleton and cell polarity, neuronal proteins, and components of signal transduction pathways mediating the hormone ecdysone. 60. Ishizuka A, Siomi MC, Siomi H: A Drosophila Fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev 2002, 16: