REVIEW (2006) 25, 6156 6162 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA Micro-RNAs (mirnas) are a class of approximately 22-nucleotide non-coding RNAs expressed in multicellular organisms.they are first transcribed in a similar manner to pre-mrnas.the transcripts then go through a series of processing steps, including endonucleolytic cleavage, nuclear export and a strand selection procedure, to yield the single-stranded mature mirna products.the transcription and processing of mirnas determines the abundance and the sequence of mature mirnas and has important implications for the function of mirnas. (2006) 25, 6156 6162. doi:10.1038/sj.onc.1209908 Keywords: micro-rna;micro-rna processing;drosha; precursor mirnas Micro-RNAs: the first impression When Ambros, Ruvkun and colleagues reported the cloning and functional analysis of the first micro-rna (mirna), lin-4, in 1993, they also provided a glimpse of things to come on the mechanism of mirna production (Lee et al., 1993;Wightman et al., 1993). The authors demonstrated that there existed two RNA species, the 22-nucleotide (nt) lin-4s and the less abundant, approximately 60 nt lin-4l. They surmised that lin-4s was the active RNA species, whereas lin-4l could be a posttranscriptional processing intermediate of lin-4s. It is not surprising that lin-4 would go through a maturation process after all, every eucaryotic RNA molecule does but the authors had the acumen to point out that the lin-4l RNA potentially forms a fold-back secondary structure, with lin-4s present in one arm of the stem. The authors also suggested that the expression of lin-4 was developmentally regulated, a feature paramount for its role in developmental timing in Caenorhabditis elegans. As our knowledge of mirnas has exploded in recent years, along with the number of mirnas identified or predicted in various genomes, the above initial observations have proved to be instrumental in guiding the research on mirna biogenesis and function. In this review, our current understanding of the mechanism of Correspondence: Dr, Department of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA. E-mail: zengx033@umn.edu mirna biogenesis is summarized, with the focus being the specificity of mirna expression. mirnas: the numbers We now know that mirnas are approximately 22-ntlong regulatory RNAs expressed in multicellular organisms (reviewed by Bartel, 2004). The sequence of mirnas is generally diverse, although a U residue is overrepresented at their 5 0 end (Lau et al., 2001). Based on sequence homology, mirnas can be grouped into sub-families, and many of them are evolutionarily conserved. For example, approximately one-third of C. elegans mirnas have vertebrate homologs (Lim et al., 2003). Many mirnas, like lin-4, have tissue-specific or developmental-stage-specific expression patterns (Feinbaum and Ambros, 1999;Reinhart et al., 2000;Lagos- Quintana et al., 2001;Lau et al., 2001;Lee and Ambros, 2001). As a result, the abundance of a particular mirna can vary greatly depending on cell type. The copy number of highly expressed mirnas may exceed 10 4 per cell, a number similar to that of many small nuclear RNAs and much higher than that of a typical mrna (Lim et al., 2003). Two approaches have been used to identify mirna genes on a large scale: the direct biochemical cloning approach and the computational biology approach. Both approaches apply what Lee et al. (1993) had described for lin-4 as an essential criterion for mirna classification: mirnas are located within stretches of sequences that are predicted by computer folding programs to form imperfect stem-loop structures, with the mature mirnas residing within one arm of the hairpin (Ambros et al., 2003a). Examples of such hairpins that encode lin-4 and mir163 in Arabidopsis thaliana are shown in Figure 1. So far, hundreds of mirnas have been identified or predicted from the genomes of plants, worms, flies, to mammals. For example, the human genome is predicted to encode as many as 1000 mirnas, approximately 3% of the total number of genes (Bentwich et al., 2005;Berezikov et al., 2005). Not all predictions have been experimentally verified: some may prove to be false positives, but many mirnas may simply have restricted expression patterns that have so far eluded detection. It is clear that mirnas are a large family of RNA molecules that can be expressed at vastly different levels spatially and temporally, which brings up an important
6157 Figure 1 Computer-predicted hairpin structures that encode mirnas. Mature mirna sequences are in red. Only one of many potential secondary structures is shown for C. elegans lin-4 (a) and for A. thaliana mir163 (b). lin-4 sequence is based on lin-4s in Lee et al. (1993). question: What is the mechanism that controls the expression of mirnas with defined 5 0 and 3 0 ends? mirnas: the transcription Transcription is a potential checkpoint in mirna expression. mirnas are initially transcribed as part of a much longer primary transcript (Lee et al., 2002; reviewed by Cullen, 2004;Kim, 2005). Several primary transcripts have been analysed in mammals, plants, worms and flies (Tam, 2001;Aukerman and Sakai, 2003; Bracht et al., 2004;Cai et al., 2004;Kurihara and Watanabe, 2004;Lee et al., 2004;Houbaviy et al., 2005; Sokol and Ambros, 2005). Full-length versions of the primary transcripts are shown to be generally quite long (>1 kb) and contain a 5 0 7-methyl guanosine cap and a 3 0 poly(a) tail. These are the characteristics of RNA polymerase II (Pol II) transcripts such as pre-mrnas. In addition, multiple studies have characterized, at least partially, the promoters that direct mirna transcription (Johnson et al., 2003;Johnston and Hobert, 2003; Cai et al., 2004;Lee et al., 2004;Fazi et al., 2005; Houbaviy et al., 2005;O Donnell et al., 2005;Sokol and Ambros, 2005;Zhao et al., 2005;Conaco et al., 2006). These promoters also bear the hallmark of Pol II promoters. For example, several tissue- or cell typespecific transcription factors are shown to regulate promoter activities, such as MyoD, Mef2 and serum responsive factor for the expression of mouse mir-1 in the heart (Zhao et al., 2005), and Twist and Mef2 for the expression of fly mir-1 (Sokol and Ambros, 2005). Available evidence, therefore, indicates that most mirnas are Pol II products, although RNA Pol III may also be involved, for example, in the transcription of several mirnas encoded by mouse gammaherpesvirus 68 (Pfeffer et al., 2005) or by adenoviruses (Andersson et al., 2005). The finding that Pol II is responsible for mirna transcription parsimoniously answers the question of why mirnas diverge in their expression levels, because Pol II promoters are highly regulated and can vary greatly in strength. A genomic analysis of mirnas has revealed that over 50% of mammalian mirnas are located within the intronic regions of annotated protein-coding or nonprotein-coding genes (Rodriguez et al., 2004). These mirnas could therefore use their host gene transcripts as carriers, although it remains possible that some are actually transcribed separately from internal promoters. Other mirnas are located in intergenic regions, apparently have their own transcriptional regulatory elements and thus constitute independent transcription units. The transcription of mirnas is reminiscent of the transcription of another class of RNAs, small nucleolar RNAs. The latter are either transcribed independently or encoded within introns of protein-coding genes. The subsequent maturation pathway for mirnas, however, is quite different from that of small nucleolar RNAs (Eliceiri, 1999). mirnas: the processing From a primary mirna transcript that potentially extends 1 kb, or even much longer, to an approximately 22 nt mature mirna, the RNA must go through a series of processing steps (reviewed by Cullen, 2004; Du and Zamore, 2005;Kim, 2005). There are some differences between the processing of animal mirnas (Figure 2a) and that of plant mirnas (Figure 2b), as noted below. Production of mirna precursors in animal cells The first step is the generation in the nucleus of an approximately 60 nt long hairpin RNA, called the precursor mirna, or pre-mirna (Figure 2a), the prototype being lin-4l observed by Lee et al. (1993). The precursor is excised from the primary transcript by an enzyme called Drosha (Lee et al., 2003). Drosha belongs to a class of RNaseIII type endonucleases (Figure 3), which produce duplex RNA products containing a 5 0 phosphate and a 3 0 -OH, with usually a 2 nt overhang at the 3 0 end. By itself, Drosha exhibits little or no enzymatic activity, as it requires a regulatory subunit called DGCR8 in humans or Pasha in flies (Denli et al., 2004;Gregory et al., 2004;Han et al., 2004;Landthaler et al., 2004). DGCR8 contains two double-stranded RNA-binding domains (Figure 3), which presumably help the catalytic Drosha subunit to recognize the correct substrates, although the structural details of how the holoenzyme interacts with RNAs remain to be determined. What RNA features does the Drosha holoenzyme (Drosha for short) recognize? In other words, what
6158 Figure 2 mirna biogenesis pathways in animals (a) and plants (b). Homologous proteins are used in both pathways and shown for comparisons in (a) and (b). The asterisks in (b) represent methylation at the 3 0 ends. Figure 3 Schematic representation of the domain structures of human Drosha, DGCR8, Dicer, TRBP and PACT. Drosha and Dicer have two RNaseIII domains, each of which makes a single cut on an RNA strand. The Dicer PAZ domain binds a 3 0 overhang of an RNA substrate. Functions of the remaining domains are mostly unknown, although the double-stranded RNA-binding domains are supposed to bind RNAs that are largely doublestranded, and the DUF283 domain of Dicer may serve a structural role (MacRae et al., 2006). distinguishes mirna-containing hairpins from so many other RNA hairpins in a cell? Both cell culture experiments and in vitro Drosha cleavage assays have shown that, for processing to occur, an extension of several base-paired residues is required beyond the final pre-mirna product (Lee et al., 2003;Zeng and Cullen, 2003). Furthermore, a flanking, single-stranded RNA is required for efficient processing in vitro (Zeng and Cullen, 2005;Han et al., 2006). At the other end of the hairpin, a large terminal loop (>10 nt) is preferred by Drosha (Zeng et al., 2005). Commonly used computer folding programs, in order to maximize the stability of RNA hairpins, tend to predict small terminal loops (e.g., 5 nt loops) for pre-mirnas, yet mutants with artificially constrained small loops are poor substrates for processing. Thus, it appears that the structure of the terminal loop region is flexible, with Drosha selecting a more open conformation. Altogether, Drosha recognizes the local structure of a relevant hairpin, but does not utilize the property of a 5 0 7-methyl guanosine cap or a 3 0 poly(a) tail. As a result, mirna processing may take place before the primary transcript is completely synthesized. Once pre-mirnas are produced, they are exported from the nucleus to the cytoplasm by Exportin5 (Exp5) and its Ran-guanosine triphosphate (GTP) cofactor (Figure 2a;Yi et al., 2003;Bohnsack et al., 2004;Lund et al., 2004). Exp5 is a member of the karyopherin family that mediates macromolecular nucleocytoplamic trafficking. Exp5 is specialized at binding to minihelixcontaining RNAs with a 3 0 overhang (Gwizdek et al., 2003), such as the human Y1 RNA, adenovirus VA1 RNA, trnas and pre-mirnas. The Exp5/Ran-GTP complex has a very high affinity for pre-mirnas, as their protein RNA interaction can be facilely demonstrated in vitro. Such a feature may endow Exp5 with a second function to sequester and protect pre-mirnas from the moment they are generated in the nucleus until they are ready for the next cleavage step in the
cytoplasm. In the cytoplasm, GTP is hydrolysed to guanosine diphosphate (GDP), and Exp5/Ran-GDP then releases its cargo. Dicer cleavage of pre-mirnas Dicer is another RNaseIII-type enzyme, and it cleaves pre-mirnas in the cytoplasm (Billy et al., 2001; Grishok et al., 2001;Hutva gner et al., 2001;Ketting et al., 2001;Provost et al., 2002;Zhang et al., 2002). Most Dicer proteins possesses an approximately 130- amino-acid-long PAZ domain (Figure 3), which preferentially binds to single-stranded 3 0 ends of doublestranded RNAs (Lingel et al., 2003;Song et al., 2003; Yan et al., 2003;Ma et al., 2004). As a pre-mirna generated by Drosha already contains a 2 nt 3 0 overhang, Dicer would recognize the 3 0 overhang via its PAZ domain and cleave the double-stranded region approximately 20 nt away, with a single catalytic center formed intramolecularly by its two RNaseIII domains (Zhang et al., 2004). The product is an mirna duplex intermediate containing approximately 2 nt 3 0 overhangs at both ends (Figure 2). A recent structural study of Dicer has confirmed that Dicer indeed acts as a molecular ruler to cleave double-stranded RNA substrates at a set distance from one end (MacRae et al., 2006). Dicer possesses a robust enzymatic activity in vitro. Nevertheless, just like DGCR8 in the case of Drosha, proteins with double-stranded RNA-binding domains, such as TRBP and PACT in humans (Figure 3), bind to Dicer and contribute to Dicer function (Chendrimada et al., 2005;Forstemann et al., 2005;Gregory et al., 2005;Haase et al., 2005;Jiang et al., 2005;Maniataki and Mourelatos, 2005;Saito et al., 2005;Lee et al., 2006). Thus, they can enhance the affinity of Dicer for RNAs and participate in the selection of mature mirna strands and/or the transfer of mirnas to their final stop, the Argonaute proteins. Selection of the mature mirna strand Dicer produces an mirna duplex intermediate, yet usually only one of the two strands can be detected in cells, heralded by the finding of Lee et al. (1993) that although lin-4l has two arms in its hairpin structure, only lin-4s from the 5 0 side was observed. How the asymmetry is achieved mechanistically is not known, although it likely involves differential binding to, and then differential retention of, the two individual RNA strands by Dicer and its associating proteins (Tomari et al., 2004). At the RNA sequence level, it appears that the strand with the less stable hydrogen bonding at its 5 0 end within the original duplex is selectively stabilized and becomes the mature mirna, whereas its complementary strand is lost (Khvorova et al., 2003;Schwarz et al., 2003). This property might explain partly why so many mirnas have a U residue at their 5 0 ends. Such mirnas have the highest chance of being selected, for a U:G base pair is less stable than a U:A pair, which in turn is less stable than a G:C pair. By eliminating half of the RNAs from the duplexes, the selection process suppresses the noise of mirna expression and reduces the number of genes whose expression could be targeted by mirna duplexes. Dicer or its complex interacts with a family of conserved proteins called Argonautes (Bernstein et al., 2001;Hammond et al., 2001;Doi et al., 2003;Tahbaz et al., 2004;Chendrimada et al., 2005;Forstemann et al., 2005;Gregory et al., 2005;Haase et al., 2005;Jiang et al., 2005;Maniataki and Mourelatos, 2005;Saito et al., 2005;Lee et al., 2006). Mature mirnas are eventually transferred to Argonaute proteins and serve as guides in RNA silencing (Caudy et al., 2002; Hutva gner and Zamore, 2002;Ishizuka et al., 2002; Mourelatos et al., 2002;Liu et al., 2004;Meister et al., 2004). In addition to being the effecters of RNA silencing, Argonaute proteins may also contribute to mirna biogenesis by selecting or binding to and subsequently stabilizing mature mirnas. mirna processing in plants Our knowledge of mirna processing in plants is mostly based on genetic analysis in A. thaliana. Many of the proteins involved in animal mirna processing have homologs in plants (Figure 2), with the exception of Drosha and DGCR8. In Arabidopsis, a Dicer-like enzyme, DCL1, appears to perform the analogous functions of both Drosha and Dicer of animal cells (Reinhart et al., 2002;Papp et al., 2003;Kurihara and Watanabe, 2004). Precursor mirnas in plants are longer, more heterogeneous in size and more difficult to detect than those in animals (Ambros et al., 2003a). Nevertheless, for at least one mirna, mir163, it has been demonstrated that DCL1 first cuts near the bottom of an extensive stem-loop structure to release a long hairpin RNA, and then the enzyme trims approximately 21 nt from the free ends progressively into the stem (Figure 2b;Kurihara and Watanabe, 2004). Because DCL1 is probably a nuclear protein, mirna duplex intermediates are also probably produced in the nucleus (Papp et al., 2003), unlike the situation in mammals. The function of plant HYL1 (Figure 2b) is likely similar to that of DGCR8, TRBP or PACT in animals (Hiraguri et al., 2005;Kurihara et al., 2006). The Exp5 homolog in plants, HST and AtRAN1, a Ran homolog, presumably export mirna duplexes to the cytoplasm, although direct biochemical evidence is lacking (Park et al., 2005). After strand selection, mirnas associate with Argonaute proteins to form gene-silencing complexes. mirnas: the modifications In Arabidopsis, the protein HEN1 methylates the ribose of the most 3 0 distal nt of an mirna, at the duplex intermediate stage (Figure 2b;Yu et al., 2005). In hen1 mutants, mirnas become uridylated at their 3 0 ends and destabilized (Li et al., 2005). Although methylation of animal mirnas has not been described, uridine addition has been associated with mrna degradation in animal cells (Shen and Goodman, 2004). 6159
6160 Another type of mirna modification is A-to-I editing by adenosine deaminases. Several studies have identified such editing within or near mature mirna sequences (Luciano et al., 2004;Pfeffer et al., 2005;Blow et al., 2006). A-to-I editing potentially affects mirna biogenesis as well as mirna function. mirna biogenesis: the big picture To conclude, we return to the question posed earlier: How can hundreds of mirnas be expressed at vastly different levels in different cells? As the transcription of mirnas is essentially the same as that of pre-mrnas, and the differential expression pattern of mirnas mirrors that of mrnas, at the first approximation, differential transcription of mirna genes is likely the main reason for the differential expression of most mirnas. If the downstream processing events were compromised in a particular cell type, the cells would make little or no mirnas at all. 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Drosha likely differentiates not only whether a transcript encodes an mirna or not but also whether a mirna primary transcript is a good substrate or a poor one. Furthermore, Drosha is a key determinant of which part of a primary transcript will become the mature mirna. The cleavage sites chosen by Drosha largely dictate where Dicer will subsequently cleave and hence, after strand selection, which RNA strand remains as the final product. Many mirnas have heterogeneous ends, probably as a result of flexible RNA-Drosha and/or RNA Dicer interactions. Understanding where Drosha and Dicer cleave is important for two main reasons. First, the cleavage sites determine the sequence of mature mirnas that are either endogenously expressed or ectopically expressed for the purpose of RNA interference. Second, the cleavage sites directly impact the function of mirnas. 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