Current Biology Vol 15 No 2 R61 man, confirming the mouse data [1]. The work with photolyase mice may therefore pave the way to our understanding of the actual causes of skin cancer in human populations. Combinations of photolyase mice lacking DNA repair ��� XP, CS or TTD mice ��� will soon be available and will help to unravel the complex, but exciting, open questions on the relationship of DNA damage and tumorigenesis. They will also help to answer the precise implications of UVB on sunlight effects, thus discriminating the genotoxicity of solar UVA irradiation, which is thought to act mainly through oxidative damage [20]. These studies will certainly be a careful alert to those people who still think of tanned skin as a healthy sign. References 1. Jans, J., Schul, W., Sert, Y.G., Rijksen, Y., Rebel, H., Eker, A.P., Nakajima, S., Steeg, H., Gruijl, F.R., Yasui, A., et al. (2005). Powerful skin cancer protection by a CPD-photolyase transgene. Curr. Biol. this issue. 2. Thompson, C.L., and Sancar, A. (2002). Photolyase/cryptochrome blue-light 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., Hoeijmakers, J.H.J., et al. (2002). Enhanced repair of cyclobutane pyrimidine dimers and improved UV resistance in photolyase transgenic mice. EMBO J. 21, 4719���4729. 4. Chigan��as, V., Batista, L.F., Brumatti, G., Amarante-Mendes, G.P., Yasui, A., and Menck, C.F. (2002). Photorepair of RNA polymerase arrest and apoptosis after ultraviolet irradiation in normal and XPB deficient rodent cells. Cell Death Differ. 9, 1099���1107. 5. You, Y.H., Lee, D.H., Yoon, J.H., Nakajima, S., Yasui, A., and Pfeifer, G. (2001). Cyclobutane pyrimidine dimers are responsible for the vast majority of mutations induced by UVB irradiation in mammalian cells. J. Biol. Chem. 276, 44688���44694. 6. Mitchell, D.L. (1988). The relative cytotoxicity of (6���4) photoproducts and cyclobutane dimers in mammalian cells. Photochem. Photobiol. 48, 51���57. 7. Zdzienicka, M.Z., Venema, J., Mitchell, D.L., van Hoffen, A., van Zeeland, A.A., Vrieling, H., Mullenders, L.H., Lohman, P.H., and Simons, J.W. (1992). (6���4) photoproducts and not cyclobutane pyrimidine dimers are the main UV- induced mutagenic lesions in Chinese hamster cells. Mutat. Res. 273, 73���83. 8. Gentil, A., Le Page, F., Margot, A., Lawrence, C.W., Borden, A., and Sarasin, A. (1996). Mutagenicity of a unique thymine-thymine dimer or thymine- thymine pyrimidine pyrimidone (6���4) photoproduct in mammalian cells. Nucleic Acids Res. 24, 1837���1840. 9. Costa, R.M., Chigan��as, V., Galhardo, R.S., Carvalho, H., and Menck, C.F. (2003). The eukaryotic nucleotide excision repair pathway. Biochimie 85, 1083���1099. 10. Riou, L., Zeng, L., Chevallier-Lagente, O., Stary, A., Nikaido, O., Taieb, A., Weeda, G., Mezzina, M., and Sarasin, A. (1999). The relative expression of mutated XPB genes results in xeroderma pigmentosum/Cockayne���s syndrome or trichothiodystrophy cellular phenotypes. Hum. Mol. Genet. 8, 1125���1133. 11. Hanawalt, P.C. (2001). Revisiting the rodent repairadox. Environ. Mol. Mutagen. 38, 89���96. 12. Lehmann, A. (2003). DNA repair-deficient diseases, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Biochimie 85, 1101���1111. 13. Asahina, H., Han, Z., Kawanishi, M., Kato, T., Ayaki, H., Todo, T., Yagi, T., Takebe, H., Ikenaga, M., and Kimura, S.H. (1999). Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells. Mutat. Res. 435, 255���262. 14. Nakajima, S., Lan, L., Kanno, S., Takao, M., Yamamoto, K., Eker, A.P., and Yasui, A. (2004). UV light-induced DNA damage and tolerance for the survival of nucleotide excision repair-deficient human cells. J. Biol. Chem. 279, 46674���46677. 15. Otoshi, E., Yagi, T., Mori, T., Matsunaga, T., Nikaido, O., Kim, S., Hitomi, K., Ikenaga, M., and Todo, T. (2000). Respective roles of cyclobutane pyrimidine dimers, (6���4) photoproducts, and minor photoproducts in ultraviolet mutagenesis of repair-deficient xeroderma pigmentosum A cells. Cancer Res. 60, 1729���1735. 16. Parris, C.N., and Kraemer, K.H. (1993). Ultraviolet-induced mutations in Cockayne syndrome cells are primarily caused by cyclobutane dimer photoproducts while repair of other photoproducts is normal. Proc. Natl. Acad. Sci. USA 90, 7260���7264. 17. Marionnet, C., Armier, J., Sarasin, A., and Stary, A. (1998). Cyclobutane pyrimidine dimers are the main mutagenic DNA photoproducts in DNA repair-deficient trichothiodystrophy cells. Cancer Res. 58, 102���108. 18. Riou, L., Eveno, E., van Hoffen, A., van Zeeland, A.A., Sarasin, A., and Mullenders, L.H. (2004). Differential repair of the two major UV-induced photolesions in trichothiodystrophy fibroblasts. Cancer Res. 64, 889���894. 19. Stege, H., Roza, L., Vink, A.A., Grewe, M., Ruzicka, T., Grether-Beck, S., and Krutmann, J. (2000). Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin. Proc. Natl. Acad. Sci. USA 97, 1790���1795. 20. Agar, N.S., Halliday, G.M., Barnetson, R.S., Ananthaswamy, H.N., Wheeler, M., and Jones, A.M. (2004). The basal layer in human squamous tumors harbors more UVA than UVB fingerprint mutations: a role for UVA in human skin carcinogenesis. Proc. Natl. Acad. Sci. USA 101, 4954���4959. Department of Microbiology, Institute of Biomedical Sciences, S��o Paulo, SP, Brazil. E-mail: kmlima@usp.br, cfmmenck@usp.br. DOI: 10.1016/j.cub.2004.12.056 Yukihide Tomari and Phillip D. Zamore MicroRNAs (miRNAs) are small RNA guides that repress the expression of their target genes. miRNAs generally have their own genes, distinct from the targets they regulate, but occasionally they cohabit with the introns or untranslated regions of genes that encode proteins. Human cells produce hundreds of distinct miRNAs, each of which is believed to act via the RNA silencing pathway to regulate mRNA stability or translation, or chromatin structure, much as small interfering RNAs do in the RNAi pathway. Regulation of gene expression by miRNAs has been proposed to be combinatorial, with different miRNAs acting together on a gene to tune its precise level of expression during development and in response to environmental stimuli. Ambros and colleagues [1] discovered the first miRNA in 1993, but miRNAs were not recognized as a new and extensive class of regulatory molecules until 2001. Hence there is great interest in how miRNAs are transcribed and processed to their mature form, how they function, and why they evolved. miRNAs are transcribed by RNA polymerase II as primary miRNAs MicroRNA Biogenesis: Drosha Can���t Cut It without a Partner The ribonuclease Drosha requires a dedicated double-stranded RNA binding protein to convert long, nuclear primary microRNA transcripts into shorter pre-microRNA stem���loops, the cytoplasmic precursors from which mature microRNAs are ultimately excised.

(pri-miRNAs) hundreds to thousands of nucleotides long [2���4]. The ribonuclease III (RNase III) enzyme Drosha cleaves the flanks of pri-miRNAs to liberate ���70 nucleotide stem���loop structures, called precursor miRNAs (pre-miRNAs). Pre- miRNAs contain the ���22 nucleotide mature miRNA in either the 5��� ��� or 3��� ��� half of their stem [5], and the pair of cuts made by Drosha establishes either the 5��� ��� or the 3��� ��� end of the mature miRNA [6]. Pre-miRNAs are moved from the nucleus to the cytoplasm by the protein Exportin 5, which recognizes the two- or three- nucleotide 3��� overhang left by Drosha at the base of the pre- miRNA stem, an end structure characteristic of RNase III cleavage [7���9]. In the cytoplasm, a second RNase III enzyme, Dicer, makes the pair of cuts that defines the other end of the miRNA, generating an siRNA-like duplex, the miR/miR* duplex. Assembly of the mature, single- stranded miRNA from the duplex into the RNA-induced silencing complex (RISC) completes miRNA biogenesis (Figure 1). Now, four laboratories [10���13] have discovered that the double- stranded RNA-binding protein known as Pasha in flies, or its ortholog DGCR8 in Caenorhabditis elegans and mammals, acts together with Drosha to convert pri-miRNA to pre-miRNA. Pasha/DGCR8 is thought to bind directly to the central region and the RNase III domains (RIIIDs) of Drosha [12]. In fact, one route to identifying Pasha/DGCR8 was its published interaction in a genome- wide yeast two-hybrid screen of Drosophila proteins [10,13]. Drosha and Pasha/DGCR8 co- purify as a 500���650 kDa nuclear complex [10���12], the microProcessor, in which pri- miRNAs are envisioned to be converted to pre-miRNAs. The microProcessor may comprise more than one of each copy of Drosha and/or Pasha/DGCR8 or it may contain proteins in addition to Drosha and Pasha/DGCR8 [12]. Like Drosha, Pasha/DGCR8 is required in vivo to convert pri- miRNA to pre-miRNA. In flies, worms, and cultured mammalian cells, reducing the level of either Drosha or Pasha/DGCR8 by RNAi led to the accumulation of pri- miRNAs and a reduction in both pre-miRNAs and mature miRNAs [10���13], underscoring the remarkable conservation of the miRNA biogenesis and RNAi machinery among metazoans. Reconstitution of pri-miRNA processing in vitro required only recombinant human Drosha and DGCR8 neither protein alone was active [11]. The finding that Drosha requires a double- stranded RNA-binding protein partner is striking, because Drosophila Dicer-2 forms a heterodimeric complex with the double-stranded RNA-binding protein R2D2, which is required for its function in RISC assembly [14], although Dicer alone suffices to convert long dsRNA into siRNAs [14,15] and pre-miRNA into miR/miR* duplexes [15]. Discovered by Zinder and colleagues in 1968, RNase III enzymes cut double-stranded RNA, using Mg2+ to facilitate catalysis. RNase III enzymes typically contain both RIIIDs and double-stranded RNA-binding domains. Class I RNase III proteins, found in bacteria and yeast, have a single RIIID. Drosha belongs to class II, and it contains two tandem RIIIDs. Dicer is a class III enzyme, with an amino- terminal helicase domain and a PAZ domain (thought to bind the single-stranded tails of siRNA duplexes) in addition to two tandem RIIIDs (Figure 2). The crystal structure of the RIIID of the Aquifex aeolicus class I RNase III revealed it to be a homodimer and suggested that the catalytic centers lie at the dimer interface. Reasoning from the Aquifex crystal structure, Dicer was originally proposed to function as a dimer, with four RIIIDs breaking four phosphodiester bonds. Subsequently, Filipowicz and colleagues [15], in a tour-de-force structure���activity study, demonstrated that both Escherichia coli RNase III (class I) and human Dicer (class III) make a pair of cuts. They showed that Dicer���s two RIIIDs form an intramolecular dimer that creates one pair of catalytic sites [15]. Kim and colleagues have now extended these studies to Drosha, a class II RNase III, demonstrating that it also contains a single processing center comprising two catalytic sites that each break one phosphodiester bond. The new data suggest that the two RIIIDs of Drosha also form an intramolecular dimer that defines the base of the pre-miRNA stem by cleaving the 5��� and 3��� sides of the pri-miRNA, leaving the approximately two nucleotide 3��� overhanging end required for recognition by Exportin 5. Thus, all RNase III enzymes likely function as intermolecular (class I) or intramolecular (classes II and III) dimers of RIIIDs and break just two phosphodiester bonds at a time. So why do RNase III, Drosha and Dicer use such different substrate RNAs, and how do they yield products so different in length and structure? Their double-stranded RNA-binding protein partners may be part of the answer (Figure 2). Recombinant human Drosha alone shows non-specific RNase activity, but the addition of Dispatch R62 Figure 1. The miRNA biogenesis pathway in animals. Mature miRNA miR/miR* duplex pri-miRNA 7mG(5��)ppp(5��)G A���AAAn-3�� Dicer RISC assembly Nucleus Cytoplasm Exportin 5 Drosha Pasha (DGCR8) pre-miRNA pre-miRNA Current Biology