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man, confirming the mouse R.S., Carvalho, H., and Menck, C.F. Res. 60, 1729–1735. (2003). The eukaryotic 16. Parris, C.N., and Kraemer, K.H. (1993). data [1]. excision repair pathway. Biochimie 85, Ultraviolet-induced mutations in The work with photolyase mice 1083–1099. Cockayne syndrome cells are primarily may therefore pave the way to our 10. Riou, L., Zeng, L., Chevallier-Lagente, O., caused by cyclobutane dimer Stary, A., Nikaido, O., Taieb, A., Weeda, photoproducts while repair of other understanding of the actual G., Mezzina, M., and Sarasin, A. (1999). photoproducts is normal. Proc. Natl. causes of skin cancer in human The relative expression of mutated XPB Acad. Sci. USA 90, 7260–7264. results in xeroderma 17. Marionnet, C., Armier, J., Sarasin, A., and populations. Combinations of pigmentosum/Cockayne’s syndrome or Stary, A. (1998). Cyclobutane pyrimidine photolyase mice lacking DNA trichothiodystrophy cellular phenotypes. dimers are the main mutagenic DNA repair — XP, CS or TTD mice — Hum. Mol. Genet. 8, 1125–1133. photoproducts in DNA repair-deficient 11. Hanawalt, P.C. (2001). Revisiting the trichothiodystrophy cells. Cancer Res. will soon be available and will help rodent repairadox. Environ. Mol. 58, 102–108. to unravel the complex, but Mutagen. 38, 89–96. 18. Riou, L., Eveno, E., van Hoffen, A., van 12. Lehmann, A. (2003). DNA repair-deficient Zeeland, A.A., Sarasin, A., and exciting, open questions on the diseases, xeroderma pigmentosum, Mullenders, L.H. (2004). Differential relationship of DNA damage and Cockayne syndrome and repair of the two major UV-induced tumorigenesis. They will also help trichothiodystrophy. Biochimie 85, photolesions in trichothiodystrophy 1101–1111. fibroblasts. Cancer Res. 64, 889–894. to answer the precise implications 13. Asahina, H., Han, Z., Kawanishi, M., 19. Stege, H., Roza, L., Vink, A.A., Grewe, of UVB on sunlight effects, thus Kato, T., Ayaki, H., Todo, T., Yagi, T., M., Ruzicka, T., Grether-Beck, S., and discriminating the genotoxicity of Takebe, H., Ikenaga, M., and Kimura, Krutmann, J. (2000). plus light S.H. (1999). Expression of a mammalian therapy to repair DNA damage in solar UVA irradiation, which is DNA photolyase confers light-dependent ultraviolet-B-irradiated human skin. Proc. thought to act mainly through repair activity and reduces mutations of Natl. Acad. Sci. USA 97, 1790–1795. UV-irradiated shuttle vectors in 20. Agar, N.S., Halliday, G.M., Barnetson, oxidative damage [20]. These xeroderma pigmentosum cells. Mutat. R.S., Ananthaswamy, H.N., Wheeler, M., studies will certainly be a careful Res. 435, 255–262. and Jones, A.M. (2004). The basal layer alert to those people who still 14. Nakajima, S., Lan, L., Kanno, S., Takao, in human squamous tumors harbors M., Yamamoto, K., Eker, A.P., and Yasui, more UVA than UVB fingerprint think of tanned skin as a healthy A. (2004). UV light-induced DNA damage mutations: a role for UVA in human skin sign. and tolerance for the survival of . Proc. Natl. Acad. Sci. nucleotide excision repair-deficient USA 101, 4954–4959. human cells. J. Biol. Chem. 279, References 46674–46677. 1. Jans, J., Schul, W., Sert, Y.G., Rijksen, 15. Otoshi, E., Yagi, T., Mori, T., Matsunaga, Department of Microbiology, Institute of Y., Rebel, H., Eker, A.P., Nakajima, S., T., Nikaido, O., Kim, S., Hitomi, K., Biomedical Sciences, São Paulo, SP, Steeg, H., Gruijl, F.R., Yasui, A., et al. Ikenaga, M., and Todo, T. (2000). Brazil. E-mail: [email protected], (2005). Powerful skin cancer protection Respective roles of cyclobutane [email protected]. by a CPD-photolyase transgene. Curr. pyrimidine dimers, (6–4) photoproducts, Biol. this issue. and minor photoproducts in ultraviolet 2. Thompson, C.L., and Sancar, A. (2002). mutagenesis of repair-deficient Photolyase/cryptochrome blue-light xeroderma pigmentosum A cells. Cancer DOI: 10.1016/j.cub.2004.12.056 photoreceptors use photon energy to repair DNA and reset the circadian clock. Oncogene 21, 9043–9056. 3. Schul, W., Jans, J., Rijksen, Y.M., Klemann, K.H., Eker, A.P., Wit, J., Nikaido, O., Nakajima, S., Yasui, A., MicroRNA Biogenesis: Hoeijmakers, J.H.J., et al. (2002). Enhanced repair of cyclobutane pyrimidine dimers and improved UV Can’t Cut It without a Partner resistance in photolyase transgenic mice. EMBO J. 21, 4719–4729. 4. Chiganças, V., Batista, L.F., Brumatti, G., The Drosha requires a dedicated double-stranded RNA Amarante-Mendes, G.P., Yasui, A., and Menck, C.F. (2002). Photorepair of RNA binding protein to convert long, nuclear primary microRNA transcripts polymerase arrest and apoptosis after into shorter pre-microRNA stem–loops, the cytoplasmic precursors ultraviolet irradiation in normal and XPB deficient rodent cells. Cell Death Differ. from which mature are ultimately excised. 9, 1099–1107. 5. You, Y.H., Lee, D.H., Yoon, J.H., Nakajima, S., Yasui, A., and Pfeifer, G. Yukihide Tomari and RNAi pathway. Regulation of (2001). Cyclobutane pyrimidine dimers Phillip D. Zamore expression by miRNAs has been are responsible for the vast majority of mutations induced by UVB irradiation in proposed to be combinatorial, mammalian cells. J. Biol. Chem. 276, MicroRNAs (miRNAs) are small with different miRNAs acting 44688–44694. RNA guides that repress the together on a gene to tune its 6. Mitchell, D.L. (1988). The relative cytotoxicity of (6–4) photoproducts and expression of their target genes. precise level of expression during cyclobutane dimers in mammalian cells. miRNAs generally have their own development and in response to Photochem. Photobiol. 48, 51–57. 7. Zdzienicka, M.Z., Venema, J., Mitchell, genes, distinct from the targets environmental stimuli. Ambros D.L., van Hoffen, A., van Zeeland, A.A., they regulate, but occasionally and colleagues [1] discovered the Vrieling, H., Mullenders, L.H., Lohman, they cohabit with the or first miRNA in 1993, but miRNAs P.H., and Simons, J.W. (1992). (6–4) photoproducts and not cyclobutane untranslated regions of genes that were not recognized as a new and pyrimidine dimers are the main UV- encode proteins. Human cells extensive class of regulatory induced mutagenic lesions in Chinese hamster cells. Mutat. Res. 273, 73–83. produce hundreds of distinct molecules until 2001. Hence there 8. Gentil, A., Le Page, F., Margot, A., miRNAs, each of which is believed is great interest in how miRNAs Lawrence, C.W., Borden, A., and Sarasin, to act via the RNA silencing are transcribed and processed to A. (1996). Mutagenicity of a unique thymine-thymine dimer or thymine- pathway to regulate mRNA their mature form, how they thymine pyrimidine pyrimidone (6–4) stability or translation, or function, and why they evolved. photoproduct in mammalian cells. Nucleic Acids Res. 24, 1837–1840. chromatin structure, much as miRNAs are transcribed by RNA 9. Costa, R.M., Chiganças, V., Galhardo, small interfering do in the polymerase II as primary miRNAs Dispatch R62

proteins, found in bacteria and Nucleus yeast, have a single RIIID. Drosha pri-miRNA pre-miRNA pre-miRNA belongs to class II, and it contains two tandem RIIIDs. is a miR/miR* Mature class III enzyme, with an amino- duplex miRNA terminal helicase domain and a PAZ domain (thought to bind the Drosha Exportin 5 Dicer RISC single-stranded tails of siRNA Pasha assembly duplexes) in addition to two (DGCR8) tandem RIIIDs (Figure 2).

7m The crystal structure of the G(5«)ppp(5«)G A…AAAn-3« Current Biology RIIID of the Aquifex aeolicus class I RNase III revealed it to be a Figure 1. The miRNA biogenesis pathway in . homodimer and suggested that the catalytic centers lie at the (pri-miRNAs) hundreds to Drosha and Pasha/DGCR8 co- dimer interface. Reasoning from thousands of long purify as a 500–650 kDa nuclear the Aquifex crystal structure, [2–4]. The ribonuclease III (RNase complex [10–12], the Dicer was originally proposed to III) enzyme Drosha cleaves the microProcessor, in which pri- function as a dimer, with four flanks of pri-miRNAs to liberate miRNAs are envisioned to be RIIIDs breaking four ∼70 nucleotide stem–loop converted to pre-miRNAs. The phosphodiester bonds. structures, called precursor microProcessor may comprise Subsequently, Filipowicz and miRNAs (pre-miRNAs). Pre- more than one of each copy of colleagues [15], in a tour-de-force miRNAs contain the ∼22 Drosha and/or Pasha/DGCR8 or it structure–activity study, nucleotide mature miRNA in either may contain proteins in addition demonstrated that both the 5′ or 3′ half of their stem [5], to Drosha and Pasha/DGCR8 [12]. Escherichia coli RNase III (class I) and the pair of cuts made by Like Drosha, Pasha/DGCR8 is and human Dicer (class III) make a Drosha establishes either the 5′ or required in vivo to convert pri- pair of cuts. They showed that the 3′ end of the mature miRNA miRNA to pre-miRNA. In flies, Dicer’s two RIIIDs form an [6]. Pre-miRNAs are moved from worms, and cultured mammalian intramolecular dimer that creates the nucleus to the cytoplasm by cells, reducing the level of either one pair of catalytic sites [15]. Kim the protein Exportin 5, which Drosha or Pasha/DGCR8 by RNAi and colleagues have now recognizes the two- or three- led to the accumulation of pri- extended these studies to Drosha, nucleotide 3′ overhang left by miRNAs and a reduction in both a class II RNase III, demonstrating Drosha at the base of the pre- pre-miRNAs and mature miRNAs that it also contains a single miRNA stem, an end structure [10–13], underscoring the processing center comprising two characteristic of RNase III remarkable conservation of the catalytic sites that each break one cleavage [7–9]. In the cytoplasm, a miRNA biogenesis and RNAi phosphodiester bond. The new second RNase III enzyme, Dicer, machinery among metazoans. data suggest that the two RIIIDs makes the pair of cuts that Reconstitution of pri-miRNA of Drosha also form an defines the other end of the processing in vitro required only intramolecular dimer that defines miRNA, generating an siRNA-like recombinant human Drosha and the base of the pre-miRNA stem duplex, the miR/miR* duplex. DGCR8; neither protein alone was by cleaving the 5′ and 3′ sides of Assembly of the mature, single- active [11]. The finding that the pri-miRNA, leaving the stranded miRNA from the duplex Drosha requires a double- approximately two nucleotide 3′ into the RNA-induced silencing stranded RNA-binding protein overhanging end required for complex (RISC) completes miRNA partner is striking, because recognition by Exportin 5. biogenesis (Figure 1). Dicer-2 forms a Thus, all RNase III Now, four laboratories [10–13] heterodimeric complex with the likely function as intermolecular have discovered that the double- double-stranded RNA-binding (class I) or intramolecular stranded RNA-binding protein protein R2D2, which is required (classes II and III) dimers of known as Pasha in flies, or its for its function in RISC assembly RIIIDs and break just two ortholog DGCR8 in Caenorhabditis [14], although Dicer alone suffices phosphodiester bonds at a time. elegans and mammals, acts to convert long dsRNA into So why do RNase III, Drosha and together with Drosha to convert siRNAs [14,15] and pre-miRNA Dicer use such different pri-miRNA to pre-miRNA. into miR/miR* duplexes [15]. substrate RNAs, and how do they Pasha/DGCR8 is thought to bind Discovered by Zinder and yield products so different in directly to the central region and colleagues in 1968, RNase III length and structure? Their the RNase III domains (RIIIDs) of enzymes cut double-stranded double-stranded RNA-binding Drosha [12]. In fact, one route to RNA, using Mg2+ to facilitate protein partners may be part of identifying Pasha/DGCR8 was its catalysis. RNase III enzymes the answer (Figure 2). published interaction in a - typically contain both RIIIDs and Recombinant human Drosha wide yeast two-hybrid screen of double-stranded RNA-binding alone shows non-specific RNase Drosophila proteins [10,13]. domains. Class I RNase III activity, but the addition of Current Biology Vol 15 No 2 R63

Ribonuclease III protein 0 200 aa E. coli RNase III A. aeolicus RNase III Class I dsRNA-binding protein partner 0 200 aa Drosophila Drosha Drosophila Pasha C. elegans Drosha C. elegans DGCR8

Class II human Drosha human DGCR8

Drosophila Dicer-1 ? Drosophila Dicer-2 Drosophila R2D2 C. elegans Dicer C. elegans RDE-4? human Dicer ?

Class III Arabidopsis DCL1 Arabidopsis HYL1? Arabidopsis DCL2 ? Arabidopsis DCL3 ?

DEAD-like helicase superfamily Helicase superfamily C-terminal domain Ribonuclease III family DUF283 Double-stranded RNA-binding motif PAZ domain Domain with two conserved Trp (W) residues (WW domain)

Current Biology

Figure 2. Ribonuclease III family proteins and their double-stranded RNA-binding protein partners. Domain structures represent the combined predictions of Pfam (http://www.sanger.ac.uk/Software/Pfam/), InterPro (http:// www.ebi.ac.uk/interpro/), PROSITE (http://www.expasy.org/prosite/) and SMART (http://smart.embl-heidelberg.de/).

DGCR8 renders it specific for pri- suggests that the double-stranded size of HYL1 plus a DCL1 protein miRNA processing [11]. This RNA-binding domain of Drosha [20]. Might HYL1 play the role of implies that Pasha/DGCR8 is a binds the portion of the double- Pasha/DGCR8 for DCL1 in plants? specificity factor that organizes stranded stem contained in the binding of Drosha on the pri- pri-miRNA but not the pre-miRNA. References 1. Lee, R.C., Feinbaum, R.L., and Ambros, miRNA, much as R2D2 has been Of course, the positions of V. (1993). The C. elegans heterochronic proposed to organize Dicer-2 Pasha/DGCR8 and the Drosha gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell binding to siRNA. How does the double-stranded RNA-binding 75, 843–854. Drosha-Pasha/DGCR8 complex domain could be reversed, with 2. Cai, X., Hagedorn, C., and Cullen, B. recognize pri-miRNA structure? In Pasha/DGCR8 positioned at the (2004). Human microRNAs are processed from capped, polyadenylated transcripts vivo and in vitro, processing pri- base of the stem and the Drosha that can also function as mRNAs. RNA miRNA into pre-miRNA requires a double-stranded RNA-binding 10, 1957–1966. 3. Lee, Y., Kim, M., Han, J., Yeom, K.H., terminal loop greater than 10 domain near the loop. Currently, Lee, S., Baek, S.H., and Kim, V.N. (2004). nucleotides [16]. The sites of we cannot distinguish between the MicroRNA genes are transcribed by RNA Drosha cleavage appear to be two models. polymerase II. EMBO J. 23, 4051–4060. 4. Parizotto, E., Dunoyer, P., Rahm, N., measured from the loop. This In plants, Dicer-like-1 (DCL1) is Himber, C., and Voinnet, O. (2004). In distance corresponds to about 22 thought to catalyze both pri- vivo investigation of the , processing, endonucleolytic activity, and base pairs — two turns of the miRNA and pre-miRNA functional relevance of the spatial RNA helix [16]. Pasha/DGCR8 processing [17,18]. DCL1 has two distribution of a plant miRNA. Genes may bind the double-stranded double-stranded RNA-binding Dev. 18, 2237–2242. 5. Lee, Y., Jeon, K., Lee, J.T., Kim, S., and stem near the loop, orienting domains, which are required for Kim, V.N. (2002). MicroRNA maturation: Drosha to bind closer to the base. accurate pri-miRNA processing, stepwise processing and subcellular localization. EMBO J. 21, 4663–4670. Thus Pasha binding may place the yet the R2D2- or Pasha/DGCR8- 6. Lee, Y., Ahn, C., Han, J., Choi, H., Kim, RIIIDs of Drosha ∼22 base pairs like double-stranded RNA-binding J., Yim, J., Lee, J., Provost, P., Radmark, from the loop, defining the ends of protein HYL1 is required for O., Kim, S., et al. (2003). The nuclear RNase III Drosha initiates microRNA the pre-miRNA. miRNA biogenesis [19,20]. processing. Nature 425, 415–419. The finding that an additional Tantalizingly, HYL1, a nuclear 7. Yi, R., Qin, Y., Macara, I.G., and Cullen, B.R. (2003). Exportin-5 mediates the helical turn at the base of the protein, is a component of a nuclear export of pre-microRNAs and stem, beyond the end of the pre- of unknown short hairpin RNAs. Genes Dev. 17, miRNA is also required in the pri- composition, but whose reported 3011–3016. 8. Bohnsack, M.T., Czaplinski, K., and miRNA for its processing [16] molecular weight is roughly the Gorlich, D. (2004). Exportin 5 is a Dispatch R64

GTP-dependent dsRNA-binding protein miRNA biogenesis. Curr. Biol. 15, through Dicer-like 1 protein functions. that mediates nuclear export of pre- 2162–2167. Proc. Natl. Acad. Sci. USA 101, miRNAs. RNA 10, 185–191. 14. Liu, Q., Rand, T.A., Kalidas, S., Du, F., 12753–12758. 9. Lund, E., Guttinger, S., Calado, A., Kim, H.E., Smith, D.P., and Wang, X. 19. Vazquez, F., Gasciolli, V., Crete, P., and Dahlberg, J.E., and Kutay, U. (2004). (2003). R2D2, a bridge between the Vaucheret, H. (2004). The nuclear Nuclear export of microRNA precursors. initiation and effector steps of the dsRNA-binding protein HYL1 is required Science 303, 95–98. Drosophila RNAi pathway. Science 301, for microRNA accumulation and plant 10. Denli, A.M., Tops, B.B., Plasterk, R.H., 1921–1925. development, but not posttranscriptional Ketting, R.F., and Hannon, G.J. (2004). 15. Zhang, H., Kolb, F., Jaskiewicz, L., transgene silencing. Curr. Biol. 14, Processing of primary microRNAs by the Westhof, E., and Filipowicz, W. (2004). 346–351. . Nature 432, Single processing center models for 20. Han, M.H., Goud, S., Song, L., and 231–235. human Dicer and bacterial RNase III. Cell Fedoroff, N. (2004). The Arabidopsis 11. Gregory, R.I., Yan, K.P., Amuthan, G., 118, 57–68. double-stranded RNA-binding protein Chendrimada, T., Doratotaj, B., Cooch, 16. Zeng, Y., Yi, R., and Cullen, B. (2005). HYL1 plays a role in microRNA-mediated N., and Shiekhattar, R. (2004). The Recognition and cleavage of primary gene regulation. Proc. Natl. Acad. Sci. Microprocessor complex mediates the microRNA precursors by the nuclear USA 101, 1093–1098. genesis of microRNAs. Nature 432, processing enzyme Drosha. EMBO J. in 235–240. press. 12. Han, J., Lee, Y., Yeom, K.-H., Kim, Y.-K., 17. Papp, I., Mette, M.F., Aufsatz, W., Department of Biochemistry and Jin, H., and Kim, V.N. (2005). The Daxinger, L., Schauer, S.E., Ray, A., van Molecular Pharmacology, University of Drosha-DGCR8 complex in primary der Winden, J., Matzke, M., and Matzke, Massachusetts Medical School, microRNA processing. Genes Dev, in A.J. (2003). Evidence for nuclear Worcester, Massachusetts 01605, USA. press. processing of plant micro RNA and short E-mail: [email protected] 13. Landthaler, M., Yalcin, A., and Tuschl, T. interfering RNA precursors. Plant (2004). The human DiGeorge syndrome Physiol. 132, 1382–1390. critical region gene 8 and its D. 18. Kurihara, Y., and Watanabe, Y. (2004). melanogaster homolog are required for Arabidopsis micro-RNA biogenesis DOI: 10.1016/j.cub.2004.12.057

β-Catenin: A Pivot between Cell are the key read-outs of canonical Wnt signaling, and are the basis Adhesion and Wnt Signalling for Wnt-induced changes in normal and malignant development [4,5]. Mutual adhesion of cells is intimately linked to Wnt signaling Armadillo/β-catenin is thus a β through a shared component: -catenin, or Armadillo in Drosophila. truly dual-function protein, β Recent work indicates how -catenin shifts from cell adhesion to Wnt encoded by a single gene in most signaling, a switch associated with epithelial–mesenchymal transitions animals and in humans. This and cancer. sharing of a critical component between two fundamental Mariann Bienz α-catenin (Figure 1). This adhesion processes — cell adhesion and function is based on a subcellular cell signaling — may reflect a need The canonical Wnt signaling pool of β-catenin that is for coordinate control between pathway is highly conserved membrane-associated and stable. them. Indeed, cell signaling is throughout the animal kingdom [1] In contrast, the signaling coupled to a loosening of and controls numerous function of β-catenin is conferred adhesion between epithelial cells developmental processes in by a soluble cytoplasmic pool that during epithelial–mesenchymal animals as distant as flies, frogs is highly unstable in the absence transitions and other and hydra. These include early of a Wnt signal, as a result of developmental processes [6]. The embryonic patterning, multiple phosphorylations in the same intrinsic link is also manifest epithelial–mesenchymal protein’s amino terminus (Figure during cancer whose progression interactions and maintenance of 2) that earmark it for proteasome- typically depends on inappropriate stem cell compartments. A key mediated degradation. This cell signaling and loss of cadherin- effector of the Wnt pathway is β- earmark depends on the mediated adhesion [7]. catenin, the Drosophila version of combined actions of the The pivot between these two which is Armadillo. Inappropriate Adenomatous polyposis coli processes appears to reside in β- activation of β-catenin in the (APC) tumor suppressor, the Axin catenin, which potentially couples intestinal epithelium, and in other scaffolding protein and two loss of cell adhesion to increased tissues, often leads to cancer [2]. serine/threonine protein kinases: Wnt signaling if diverted from the The β-catenin protein was glycogen synthase kinase 3β plasma membrane to the nucleus. initially discovered for its role in (GSK3) and its priming kinase, Indeed, although the two β- cell adhesion [3]. As a component casein kinase 1. Upon Wnt catenin pools are normally well of adherens junctions, it promotes signaling, GSK3 is inhibited; as a buffered and functionally cell adhesion by binding to the consequence, unphosphorylated separated from each other, intracellular domain of the (‘activated’) β-catenin experimental manipulations of the transmembrane protein cadherin, a accumulates, and promotes the levels of one pool can affect the Ca2+-dependent homotypic transcription of Wnt target genes function of the other under some adhesion molecule, and linking by binding to TCF transcription circumstances (reviewed in [8]). cadherin to the actin cytoskeleton factors in the nucleus (Figure 1) The question arises whether there through the adaptor protein [2]. These transcriptional changes are control switches that flip the