Principles of Micro-RNA Production and Maturation

Y Zeng

Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA

Micro-RNAs (miRNAs) are a class of approximately miRNA biogenesis is summarized, with the focus being 22-nucleotide non-coding RNAs expressed in multicellular the specificity of miRNA expression. 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 miRNAs: the numbers the single-stranded mature miRNA products.The transcription and processing of miRNAs determines the We now know that miRNAs are approximately 22-nt- abundance and the sequence of mature miRNAs and has long regulatory RNAs expressed in multicellular organ- important implications for the function of miRNAs. isms (reviewed by Bartel, 2004). The sequence of Oncogene (2006) 25, 6156–6162. doi:10.1038/sj.onc.1209908 miRNAs is generally diverse, although a U residue is overrepresented at their 50 end (Lau et al., 2001). Based Keywords: micro-RNA;micro-RNA processing;Drosha; on sequence homology, miRNAs can be grouped into precursor miRNAs 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 (Fein- Micro-RNAs: the first impression baum and Ambros, 1999;Reinhart et al., 2000;Lagos- Quintana et al., 2001;Lau et al., 2001;Lee and Ambros, When Ambros, Ruvkun and colleagues reported the 2001). As a result, the abundance of a particular cloning and functional analysis of the first micro-RNA miRNA can vary greatly depending on cell type. The (miRNA), lin-4, in 1993, they also provided a glimpse of copy number of highly expressed miRNAs may exceed 4 things to come on the mechanism of miRNA production 10 per cell, a number similar to that of many small (Lee et al., 1993;Wightman et al., 1993). The authors nuclear RNAs and much higher than that of a typical demonstrated that there existed two RNA species, the mRNA (Lim et al., 2003). 22-nucleotide (nt) lin-4S and the less abundant, approxi- Two approaches have been used to identify miRNA mately 60 nt lin-4L. They surmised that lin-4S was the genes on a large scale: the direct biochemical cloning active RNA species, whereas lin-4L could be a post- approach and the computational biology approach. transcriptional processing intermediate of lin-4S. It is Both approaches apply what Lee et al. (1993) had not surprising that lin-4 would go through a maturation described for lin-4 as an essential criterion for miRNA process – after all, every eucaryotic RNA molecule does classification: miRNAs are located within stretches of – but the authors had the acumen to point out that the sequences that are predicted by computer folding lin-4L RNA potentially forms a fold-back secondary programs to form imperfect stem-loop structures, with structure, with lin-4S present in one arm of the stem. the mature miRNAs residing within one arm of the The authors also suggested that the expression of lin-4 hairpin (Ambros et al., 2003a). Examples of such was developmentally regulated, a feature paramount for hairpins that encode lin-4 and miR163 in Arabidopsis its role in developmental timing in Caenorhabditis thaliana are shown in Figure 1. So far, hundreds of elegans. miRNAs have been identified or predicted from the As our knowledge of miRNAs has exploded in recent genomes of plants, worms, flies, to mammals. For years, along with the number of miRNAs identified or example, the human genome is predicted to encode as predicted in various genomes, the above initial observa- many as 1000 miRNAs, approximately 3% of the total tions have proved to be instrumental in guiding the number of genes (Bentwich et al., 2005;Berezikov et al., research on miRNA biogenesis and function. In this 2005). Not all predictions have been experimentally review, our current understanding of the mechanism of 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 Correspondence: Dr Y Zeng, Department of Pharmacology, Uni- versity of Minnesota, 6-120 Jackson Hall, 321 Church Street SE, that miRNAs are a large family of RNA molecules Minneapolis, MN 55455, USA. that can be expressed at vastly different levels spatially E-mail: [email protected] and temporally, which brings up an important Principles of micro-RNA production and maturation Y Zeng 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 as carriers, although it remains possible that some are expression of miRNAs with defined 50 and 30 ends? actually transcribed separately from internal promoters. Other miRNAs are located in intergenic regions, apparently have their own transcriptional regulatory miRNAs: the transcription elements and thus constitute independent transcription units. The transcription of miRNAs is reminiscent of the Transcription is a potential checkpoint in miRNA transcription of another class of RNAs, small nucleolar expression. miRNAs are initially transcribed as part of RNAs. The latter are either transcribed independently a much longer primary transcript (Lee et al., 2002; or encoded within introns of protein-coding genes. The reviewed by Cullen, 2004;Kim, 2005). Several primary subsequent maturation pathway for miRNAs, however, transcripts have been analysed in mammals, plants, is quite different from that of small nucleolar RNAs worms and flies (Tam, 2001;Aukerman and Sakai, 2003; (Eliceiri, 1999). 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 miRNAs: the processing primary transcripts are shown to be generally quite long (>1 kb) and contain a 50 7-methyl guanosine cap and a From a primary miRNA transcript that potentially 30 poly(A) tail. These are the characteristics of RNA extends 1 kb, or even much longer, to an approximately polymerase II (Pol II) transcripts such as pre-mRNAs. 22 nt mature miRNA, the RNA must go through a In addition, multiple studies have characterized, at least series of processing steps (reviewed by Cullen, 2004; partially, the promoters that direct miRNA transcrip- Du and Zamore, 2005;Kim, 2005). There are some tion (Johnson et al., 2003;Johnston and Hobert, 2003; differences between the processing of animal miRNAs Cai et al., 2004;Lee et al., 2004;Fazi et al., 2005; (Figure 2a) and that of plant miRNAs (Figure 2b), as Houbaviy et al., 2005;O’Donnell et al., 2005;Sokol and noted below. Ambros, 2005;Zhao et al., 2005;Conaco et al., 2006). These promoters also bear the hallmark of Pol II Production of miRNA precursors in animal cells promoters. For example, several tissue- or cell type- The first step is the generation in the nucleus of an specific transcription factors are shown to regulate approximately 60 nt long hairpin RNA, called the promoter activities, such as MyoD, Mef2 and serum precursor miRNA, or pre-miRNA (Figure 2a), responsive factor for the expression of mouse miR-1 in the prototype being lin-4L observed by Lee et al. the heart (Zhao et al., 2005), and Twist and Mef2 for the (1993). The precursor is excised from the primary expression of fly miR-1 (Sokol and Ambros, 2005). transcript by an enzyme called Drosha (Lee et al., Available evidence, therefore, indicates that most 2003). Drosha belongs to a class of RNaseIII type miRNAs are Pol II products, although RNA Pol III endonucleases (Figure 3), which produce duplex RNA may also be involved, for example, in the transcription products containing a 50 phosphate and a 30-OH, with of several miRNAs encoded by mouse gammaherpes- usually a 2 nt overhang at the 30 end. By itself, Drosha virus 68 (Pfeffer et al., 2005) or by adenoviruses exhibits little or no enzymatic activity, as it requires a (Andersson et al., 2005). The finding that Pol II is regulatory subunit called DGCR8 in humans or Pasha responsible for miRNA transcription parsimoniously in flies (Denli et al., 2004;Gregory et al., 2004;Han answers the question of why miRNAs diverge in their et al., 2004;Landthaler et al., 2004). DGCR8 contains expression levels, because Pol II promoters are highly two double-stranded RNA-binding domains (Figure 3), regulated and can vary greatly in strength. which presumably help the catalytic Drosha subunit to A genomic analysis of miRNAs has revealed that over recognize the correct substrates, although the structural 50% of mammalian miRNAs are located within the details of how the holoenzyme interacts with RNAs intronic regions of annotated protein-coding or non- remain to be determined. protein-coding genes (Rodriguez et al., 2004). These What RNA features does the Drosha holoenzyme miRNAs could therefore use their host gene transcripts (Drosha for short) recognize? In other words, what

Oncogene Principles of micro-RNA production and maturation Y Zeng 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 30 ends.

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 50 7-methyl guanosine cap or a 30 Figure 3 Schematic representation of the domain structures of poly(A) tail. As a result, miRNA processing may take human Drosha, DGCR8, Dicer, TRBP and PACT. Drosha and place before the primary transcript is completely Dicer have two RNaseIII domains, each of which makes a single synthesized. cut on an RNA strand. The Dicer PAZ domain binds a 30 overhang of an RNA substrate. Functions of the remaining domains are Once pre-miRNAs are produced, they are exported mostly unknown, although the double-stranded RNA-binding from the nucleus to the cytoplasm by Exportin5 (Exp5) domains are supposed to bind RNAs that are largely double- and its Ran-guanosine triphosphate (GTP) cofactor stranded, and the DUF283 domain of Dicer may serve a structural (Figure 2a;Yi et al., 2003;Bohnsack et al., 2004;Lund role (MacRae et al., 2006). et al., 2004). Exp5 is a member of the karyopherin family that mediates macromolecular nucleocytoplamic trafficking. Exp5 is specialized at binding to minihelix- distinguishes miRNA-containing hairpins from so containing RNAs with a 30 overhang (Gwizdek et al., many other RNA hairpins in a cell? Both cell culture 2003), such as the human Y1 RNA, adenovirus VA1 experiments and in vitro Drosha cleavage assays have RNA, tRNAs and pre-miRNAs. The Exp5/Ran-GTP shown that, for processing to occur, an extension of complex has a very high affinity for pre-miRNAs, as several base-paired residues is required beyond the final their protein–RNA interaction can be facilely demon- pre-miRNA product (Lee et al., 2003;Zeng and Cullen, strated in vitro. Such a feature may endow Exp5 with a 2003). Furthermore, a flanking, single-stranded RNA is second function to sequester and protect pre-miRNAs required for efficient processing in vitro (Zeng and from the moment they are generated in the nucleus until Cullen, 2005;Han et al., 2006). At the other end of the they are ready for the next cleavage step in the

Oncogene Principles of micro-RNA production and maturation Y Zeng 6159 cytoplasm. In the cytoplasm, GTP is hydrolysed to suppresses the ‘noise’ of miRNA expression and reduces guanosine diphosphate (GDP), and Exp5/Ran-GDP the number of genes whose expression could be targeted then releases its cargo. by miRNA duplexes. Dicer or its complex interacts with a family of conserved proteins called Argonautes (Bernstein et al., Dicer cleavage of pre-miRNAs 2001;Hammond et al., 2001;Doi et al., 2003;Tahbaz Dicer is another RNaseIII-type enzyme, and it cleaves et al., 2004;Chendrimada et al., 2005;Forstemann et al., pre-miRNAs in the cytoplasm (Billy et al., 2001; 2005;Gregory et al., 2005;Haase et al., 2005;Jiang Grishok et al., 2001;Hutva ´ gner et al., 2001;Ketting et al., 2005;Maniataki and Mourelatos, 2005;Saito et al., 2001;Provost et al., 2002;Zhang et al., 2002). et al., 2005;Lee et al., 2006). Mature miRNAs are Most Dicer proteins possesses an approximately 130- eventually transferred to Argonaute proteins and serve amino-acid-long PAZ domain (Figure 3), which pre- as guides in RNA silencing (Caudy et al., 2002; ferentially binds to single-stranded 30 ends of double- Hutva´ gner and Zamore, 2002;Ishizuka et al., 2002; stranded RNAs (Lingel et al., 2003;Song et al., 2003; Mourelatos et al., 2002;Liu et al., 2004;Meister et al., Yan et al., 2003;Ma et al., 2004). As a pre-miRNA 2004). In addition to being the effecters of RNA generated by Drosha already contains a 2 nt 30 over- silencing, Argonaute proteins may also contribute to hang, Dicer would recognize the 30 overhang via its PAZ miRNA biogenesis by selecting or binding to and domain and cleave the double-stranded region approxi- subsequently stabilizing mature miRNAs. mately 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 miRNA processing in plants intermediate containing approximately 2 nt 30 overhangs Our knowledge of miRNA processing in plants is mostly at both ends (Figure 2). A recent structural study of based on genetic analysis in A. thaliana. Many of the Dicer has confirmed that Dicer indeed acts as a proteins involved in animal miRNA processing have molecular ruler to cleave double-stranded RNA sub- homologs in plants (Figure 2), with the exception of strates at a set distance from one end (MacRae et al., Drosha and DGCR8. In Arabidopsis, a Dicer-like 2006). enzyme, DCL1, appears to perform the analogous Dicer possesses a robust enzymatic activity in vitro. functions of both Drosha and Dicer of animal cells Nevertheless, just like DGCR8 in the case of Drosha, (Reinhart et al., 2002;Papp et al., 2003;Kurihara and proteins with double-stranded RNA-binding domains, Watanabe, 2004). Precursor miRNAs in plants are such as TRBP and PACT in humans (Figure 3), bind to longer, more heterogeneous in size and more difficult Dicer and contribute to Dicer function (Chendrimada to detect than those in animals (Ambros et al., 2003a). et al., 2005;Forstemann et al., 2005;Gregory et al., Nevertheless, for at least one miRNA, miR163, it has 2005;Haase et al., 2005;Jiang et al., 2005;Maniataki been demonstrated that DCL1 first cuts near the bottom and Mourelatos, 2005;Saito et al., 2005;Lee et al., of an extensive stem-loop structure to release a long 2006). Thus, they can enhance the affinity of Dicer for hairpin RNA, and then the enzyme trims approximately RNAs and participate in the selection of mature 21 nt from the free ends progressively into the stem miRNA strands and/or the transfer of miRNAs to their (Figure 2b;Kurihara and Watanabe, 2004). Because final stop, the Argonaute proteins. 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 Selection of the mature miRNA strand function of plant HYL1 (Figure 2b) is likely similar to Dicer produces an miRNA duplex intermediate, yet that of DGCR8, TRBP or PACT in animals (Hiraguri usually only one of the two strands can be detected in et al., 2005;Kurihara et al., 2006). The Exp5 homolog in et al cells, heralded by the finding of Lee . (1993) that plants, HST and AtRAN1, a Ran homolog, presumably although lin-4L has two arms in its hairpin structure, export miRNA duplexes to the cytoplasm, although only lin-4S from the 50 side was observed. How the direct biochemical evidence is lacking (Park et al., asymmetry is achieved mechanistically is not known, 2005). After strand selection, miRNAs associate with although it likely involves differential binding to, and Argonaute proteins to form gene-silencing complexes. 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 50 miRNAs: the modifications end within the original duplex is selectively stabilized and becomes the mature miRNA, whereas its comple- In Arabidopsis, the protein HEN1 methylates the ribose mentary strand is lost (Khvorova et al., 2003;Schwarz of the most 30 distal nt of an miRNA, at the duplex et al., 2003). This property might explain partly why so intermediate stage (Figure 2b;Yu et al., 2005). In hen1 many miRNAs have a U residue at their 50 ends. Such mutants, miRNAs become uridylated at their 30 ends miRNAs have the highest chance of being selected, for a and destabilized (Li et al., 2005). Although methylation U:G base pair is less stable than a U:A pair, which in of animal miRNAs has not been described, uridine turn is less stable than a G:C pair. By eliminating half of addition has been associated with mRNA degradation the RNAs from the duplexes, the selection process in animal cells (Shen and Goodman, 2004).

Oncogene Principles of micro-RNA production and maturation Y Zeng 6160 Another type of miRNA modification is A-to-I being another checkpoint in animal cells. Drosha likely editing by adenosine deaminases. Several studies have differentiates not only whether a transcript encodes an identified such editing within or near mature miRNA miRNA or not but also whether a miRNA primary sequences (Luciano et al., 2004;Pfeffer et al., 2005;Blow transcript is a ‘good’ substrate or a ‘poor’ one. et al., 2006). A-to-I editing potentially affects miRNA Furthermore, Drosha is a key determinant of which biogenesis as well as miRNA function. 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, miRNA biogenesis: the big picture after strand selection, which RNA strand remains as the final product. Many miRNAs have heterogeneous ends, To conclude, we return to the question posed earlier: probably as a result of flexible RNA-Drosha and/or How can hundreds of miRNAs be expressed at vastly RNA–Dicer interactions. Understanding where Drosha different levels in different cells? As the transcription of and Dicer cleave is important for two main reasons. miRNAs is essentially the same as that of pre-mRNAs, First, the cleavage sites determine the sequence of and the differential expression pattern of miRNAs mature miRNAs that are either endogenously expressed mirrors that of mRNAs, at the first approximation, or ectopically expressed for the purpose of RNA differential transcription of miRNA genes is likely the interference. Second, the cleavage sites directly impact main reason for the differential expression of most the function of miRNAs. It has been shown that 0 miRNAs. If the downstream processing events were sequences in the 5 half of miRNAs, especially from compromised in a particular cell type, the cells would the No. 2 to No. 7 positions (No. 1 being over- make little or no miRNAs at all. Instead, different cell represented by a U), are critical for target recognition types produce some but not the entire miRNA (Doench and Sharp, 2004;Mallory et al., 2004; repertoire encoded by the same genome. On the other Brennecke et al., 2005;Krek et al., 2005;Lai et al., hand, one can imagine that the processing of a small 2005;Lewis et al., 2005). Prediction of miRNAs by number of miRNAs may be under the control of specific current computational methods or detection of miRNAs RNA-binding proteins. These proteins can block or by Northern blot and microarray analyses cannot allow processing until an appropriate time, or even pinpoint the ends of miRNAs. To appreciate fully and modulate cleavage sites to influence strand selection. accurately the target genes whose expression is regulated Several miRNAs, for example, miR-60 and miR-38 in C. by miRNAs, one needs to pay attention to how elegans, may fit such a description, as the precursors of miRNAs are processed in vivo. these miRNAs are more consistently detected than the mature miRNAs (Lee and Ambros, 2001;Ambros et al., Acknowledgements 2003b). Even with the same level of transcription, miRNA I thank B Cullen for comments and E Wessel for help in production can still vary, with processing by Drosha preparing the figures.

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