Discovery. The discovery of miRNAs as endogenous regulators of gene expression was one of the major advances in biology. The first miRNA lin-4 was identified in 1993 in C. elegans as a regulator of developmental timing, and was found to repress the translation of its target mRNA lin-14, to which it base-paired with partial sequence complementarity (Lee et al., 1993; Wightman et al., 1993). lin-4 was perhaps initially thought of as an oddity of the worm, as no clear homologs of lin-4 were found in insects or mammals. The discovery of a second miRNA, let-7, that has homologs in all animal lineages with bilateral symmetry, led to the realization that miRNAs are common regulators of gene expression in animals (Pasquinelli et al., 2000; Reinhart et al., 2000). In 2001, three groups reported the cloning of many miRNAs from C. elegans, Drosophila and humans (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). In 2002, efforts to clone small RNAs from Arabidopsis by several groups led to the first discovery of miRNAs from plants (Llave et al., 2002a; Park et al., 2002; Reinhart et al., 2002). By now, miRNAs have been found either by homology or by cloning from plants representing many major lineages, such as green algae (Molnar et al., 2007; Zhao et al., 2007), mosses (Axtell and Bartel, 2005; Talmor-Neiman et al., 2006a,b; Axtell et al., 2007), ferns (Axtell and Bartel, 2005), gymnosperms (Lu et al., 2007; Morin et al., 2008), monocots (Sunkar et al., 2005; Yao et al., 2007) and dicots (Barakat et al., 2007a,b; Sunkar and Jagadeeswaran, 2008). The discovery of miRNAs relied mainly on size fractionation to isolate small RNAs from total RNAs, ligation of the small RNAs to 5′ and 3′ adaptors, and RT-PCR of the ligation products to generate a small RNA library, which is then sequenced. Initial efforts towards miRNA discovery relied on traditional Sanger sequencing, and resulted in the identification of mostly abundant miRNA species. The adaptation of high-throughput sequencing technologies to small RNA discovery, first performed in plants (Lu et al., 2005), greatly enriched the known repertoire of small RNAs, including miRNAs, not only in plants but also in animals. The combination of high-throughput sequencing with mutants defective in endogenous siRNA biogenesis further allowed the detection of rare miRNAs (Lu et al., 2006). A few years after the Arabidopsis genome was sequenced, a new class of regulatory genes hidden in the genome was finally unveiled. Eight years since their first discovery, plant miRNAs are now widely recognized as major players in gene regulation that impact almost all aspects of plant biology.
Biogenesis. Molecular genetic studies in Arabidopsis established the major framework of miRNA biogenesis (Figure 2). A mature miRNA lies in one arm of the stem of a hairpin RNA precursor, which is named a pre-miRNA. The hairpin RNA tends to reside in a larger RNA (as revealed by expressed sequence tags, ESTs), termed a pri-miRNA. The fact that the RNase III enzyme Dicer generates the lin-4 and let-7 miRNAs in C. elegans (Grishok et al., 2001; Hutvagner et al., 2001; Ketting et al., 2001) prompted two groups to investigate the role of DCL1, one of the four Arabidopsis homologs of Dicer, in miRNA biogenesis. The weak dcl1-9 allele was shown to have reduced accumulation of most tested miRNAs, indicating that DCL1 is a major miRNA Dicer in plants (Park et al., 2002; Reinhart et al., 2002). Whereas the C. elegans dcr-1 mutant resulted in reduced miRNA accumulation, accompanied by over-accumulation of pre-miRNAs, pre-miRNAs were not readily detectable by northern blotting in the Arabidopsis dcl1-9 mutant. In fact, dcl1 mutants had reduced levels of pre-miRNAs and increased levels of pri-miRNAs (Kurihara and Watanabe, 2004; Kurihara et al., 2006), consistent with a role of DCL1 in the processing of pri-miRNAs to pre-miRNAs, as well as pre-miRNAs to miRNA/miRNA* duplexes. Although DCL1 is the major DICER-LIKE protein that produces miRNAs in Arabidopsis, DCL4 also generates a few miRNAs that are derived from precursors with long double-stranded regions (Rajagopalan et al., 2006).
Figure 2. Three distinct endogenous small RNA pathways. (a) microRNA (miRNA) metabolism. A primary miRNA (pri-miRNA) is processed into the pre-miRNA, which is furthered processed into the miRNA/miRNA* duplex. This duplex undergoes 2′-O-methylation by the small RNA methyltransferases HEN1. One strand is bound by AGO1. The mature miRNAs are turned over by the SDN1 family of small RNA exonulceases. (b) The biogenesis of trans-acting small interfering RNAs (ta-siRNAs). A non-coding ta-siRNA-generating region (TAS) is transcribed into a single-stranded RNA, which is targeted by an miRNA for cleavage. After cleavage, one of the fragments is copied into double-stranded RNA (dsRNA) by RDR6. The dsRNA is processed into phased siRNAs by DCL4. Some of the siRNAs are bound by AGO1 and regulate other mRNAs in trans, as do miRNAs. (c) Biogenesis and function of heterochromatic siRNAs. A heterochromatic locus is presumably transcribed by Pol IV into a single-stranded RNA, which is turned into dsRNA by RDR2. The dsRNA is processed into 24-nt siRNAs, which are bound by AGO4. The AGO4/siRNA complex is attracted to the homologous DNA locus by transcripts generated by Pol V, and recruits DNA and histone modification machinery to result in heterochromatin formation. CLASSY1 and DRD1 are SNF2-like, putative chromatin remodeling proteins that act together with Pol IV and Pol V, respectively. DMS3 and KTF1 are also required for recruiting AGO4 to genomic loci.
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Other genes in miRNA biogenesis were soon identified primarily based on the similar developmental defects exhibited by mutants in these genes and dcl1 mutants. HYL1, a protein with dsRNA binding domains, and SE, a zinc-finger protein, were found to be required for miRNA biogenesis, because mutants in these genes had lower levels of most miRNAs (Han et al., 2004; Vazquez et al., 2004a; Lobbes et al., 2006; Yang et al., 2006a). The reduced accumulation of pre-miRNAs and/or over-accumulation of pri-miRNAs in these mutants placed the two genes in the pri-miRNA-to-pre-miRNA processing step, together with DCL1 (Kurihara et al., 2006; Lobbes et al., 2006; Yang et al., 2006a). The two proteins were then shown to interact with each other and with DCL1 in vitro, and to co-localize with DCL1 in vivo in so-called nuclear D-bodies, presumably sites of miRNA biogenesis (Hiraguri et al., 2005; Kurihara et al., 2006; Lobbes et al., 2006; Yang et al., 2006a; Song et al., 2007). In vitro processing assays showed that the two proteins determine the precision of excision of miRNAs by DCL1 (Dong et al., 2008).
Which polymerase generates the pri-miRNAs? ESTs from a few MIR genes indicated that pri-miRNAs were spliced and polyadenlyated, implicating Pol II in the transcription of MIR genes (Aukerman and Sakai, 2003; Kurihara and Watanabe, 2004). A comprehensive analysis of transcription start sites of numerous MIR genes revealed the presence of TATA boxes in their promoters, and indicated that Pol II transcribes MIR genes (Xie et al., 2005a). This implies that MIR genes can be subjected to transcriptional regulation, as are protein-coding genes. Indeed, many miRNAs exhibit temporally or spatially regulated patterns of expression, or accumulate in response to environmental stimuli. Mutants in the two nuclear cap binding complex (CBC) genes, CBP20 and CBP80 (also known as ABH1; Hugouvieux et al., 2001) exhibit phenotypes similar to weak se alleles in terms of leaf serration, and have reduced levels of some but not all miRNAs (Gregory et al., 2008; Laubinger et al., 2008). It was not until recently that ARS2, the SE homolog in animals, was shown to act in miRNA biogenesis in Drosophila and mice (Gruber et al., 2009; Sabin et al., 2009). The studies also revealed the physical interaction between ARS2 and CBC. Therefore, the CBPs may link pri-miRNA transcription to its processing.
After DCL1-mediated processing of pre-miRNAs to generate miRNA/miRNA* duplexes, the next step in miRNA biogenesis is methylation, a step that was completely unexpected because of a lack of a parallel process in animal miRNA biogenesis. In the course of studying floral morphogenesis, my group isolated mutants in the HEN1 gene that promotes the development of reproductive organs in the flower (Chen et al., 2002). The phenotypic similarities between hen1 and dcl1-9 plants prompted us to test whether HEN1 played a role in miRNA biogenesis. Indeed, we found that miRNAs were reduced in abundance in hen1 mutants (Park et al., 2002). Next, the presence of a methyltransferase domain in the HEN1 protein prompted us to test whether HEN1 is an miRNA methyltransferase. In vitro methyltransferase reactions with various known intermediates in miRNA biogenesis as substrates revealed that HEN1 was a methyltransferase acting only on miRNA/miRNA* duplexes, in a sequence-independent but structure-dependent manner (Yu et al., 2005). HEN1 requires the presence of both the 2′ and 3′ hydroxyls on the 3′ terminal ribose and a 2-nt 3′ overhang (features of Dicer products), and prefers 21–24-nt duplexes (sizes of products of the four Arabidopsis DCL proteins) for its activities (Yang et al., 2006b). Biochemical studies pinpointed the 2′ OH on the 3′ terminal ribose as the site of methylation (Yang et al., 2006b). A recent study solved the structure of the HEN1 protein and shed light on the structural basis for substrate recognition by HEN1 (Huang et al., 2009b). The structural studies also revealed a novel Mg2+-dependent enzymatic mechanism, not found in known RNA methyltransferases, that could account for the 2′ OH-specific methylation.
The presence of a methyl group on plant miRNAs was confirmed by β-elimination reactions that require the presence of both the 2′ and 3′ hydroxyl groups on the 3′ terminal ribose, and by mass spectrometry analysis of a purified plant miRNA species (Yu et al., 2005). It is now established that plant miRNAs carry a 2′-O-methyl group on the 3′ terminal ribose. Most animal miRNAs are not methylated, but siRNAs and piwi-interacting RNAs are 2′-O-methylated in animals by HEN1 homologs (Kim et al., 2009).
The 2′-O-methyl group appears to protect miRNAs from 3′ exonucleolytic degradation and 3′ uridylation, a process in which a short U-rich tail is added to unmethylated miRNAs (Li et al., 2005). The function of uridylation is unknown, but is likely to cause instability of miRNAs. Tailing of miRNAs or other types of small RNAs with Us or As has also been found in animals (Kim et al., 2009; van Wolfswinkel et al., 2009). We suspect that tailing is a general strategy in the regulation of miRNA stability. Our recent study identified the SDN family of exonucleases that preferentially degrades single-stranded small RNAs in vitro, and limits the abundance of miRNAs in vivo (Ramachandran and Chen, 2008). It is not yet known whether the SDN proteins preferentially degrade U-tailed or non-tailed miRNAs.
The nuclear localization of DCL1 suggests that the two processing events leading to the production of miRNA/miRNA* duplexes occur in the nucleus (Papp et al., 2003). The nuclear processing and the cytoplasmic actions of miRNAs imply an export step in miRNA biogenesis. HASTY, a homolog of the mammalian pre-miRNA export factor exportin 5, is likely to export miRNA/miRNA* duplexes or miRISCs to the cytoplasm (Park et al., 2005).
The final step in miRNA biogenesis is the incorporation of the miRNA strand into AGO1, one of 10 Arabidopsis argonaute proteins. AGO1 was first characterized for its roles in plant development, especially leaf polarity specification (Bohmert et al., 1998). Mechanistic insights into the molecular functions of argonaute proteins were provided by structural studies, which revealed that the conserved piwi domain adopted an RNase H-fold, and biochemical studies, which demonstrated that mammalian AGO2 exhibited small RNA-guided endonucleolytic activity against target mRNAs (Liu et al., 2004; Song et al., 2004). AGO1 from Arabidopsis was shown to bind miRNAs and execute the cleavage of target mRNAs (Baumberger and Baulcombe, 2005; Qi et al., 2005). In the 10-member argonaute family in Arabidopsis, AGO10 is the most closely related to AGO1, and has been implicated in the translational repression of miRNA targets (Brodersen et al., 2008). It has not been shown yet whether AGO10 associates with miRNAs. Two other genes, VCS, a member of the mRNA decapping complex, and KTN1, a subunit of the microtubule-severing enzyme KATANIN, were found to be required for miRNA-mediated translational repression of target mRNAs (Brodersen et al., 2008).
Function. Studies with the first miRNA lin-4 suggested that it inhibits the translation of its target mRNA lin-14 (Wightman et al., 1993; Olsen and Ambros, 1999). This conclusion was based on the disproportionate effects on the lin-14 protein versus mRNA levels caused by mutations in the miRNA. Animal miRNAs were later found to also cause the degradation of their target mRNAs, via decapping and/or deadenylation (Bagga et al., 2005; Behm-Ansmant et al., 2006; Eulalio et al., 2009). Although the mechanisms of action of animal miRNAs are still controversial, it is clear that the great majority of animal miRNAs pair with their target mRNAs with a central bulge, which would prevent the cleavage of the mRNAs.
When plant miRNAs were first identified, it was noted that plant miRNAs had extensive sequence complementarity to their potential targets (Llave et al., 2002a; Park et al., 2002; Reinhart et al., 2002). Soon afterwards, it was demonstrated that miR171 could lead to the cleavage of its target mRNA (Llave et al., 2002b). The 5′ end of the 3′ cleavage product of the miR171 target mRNA mapped to the nucleotide opposite the 10th nucleotide from the 5′ end of miR171 in the miR171/mRNA duplex, as determined by 5′ RACE PCR, a method that has since been widely adopted for the confirmation of miRNA targets in plants. That cleavage is not the only mode of action of plant miRNAs was realized when the effects of miR172, which targets AP2 and related genes, were analyzed (Aukerman and Sakai, 2003; Chen, 2004). Over-expression of miR172 led to phenotypes resembling ap2 loss-of-function mutants. Consistent with the phenotypic outcome, AP2 protein levels were reduced; however, AP2 mRNA levels were unaffected. Similarly, transgenic lines expressing AP2 or a miR172-resistant version of AP2 could have similar levels of the AP2 mRNA but drastically different levels of the AP2 protein, as well as different phenotypic outcomes. These observations led to the conclusion that miR172 could inhibit the translation of AP2 mRNA. miR156/157, which targets the SPL family of transcription factor genes, and whose target sites in some of the genes reside in the 3′ untranslated regions, was also found to disproportionately affect the mRNA and protein levels of its target genes (Gandikota et al., 2007). A more extensive survey of many miRNA/target pairs found widespread translational repression by plant miRNAs (Brodersen et al., 2008). Therefore, plant miRNAs, as well as animal miRNAs, regulate their target mRNAs through both mRNA degradation and translational inhibition. Intriguingly, a study conducted on miR398 and its two targets encoding copper superoxide dismutase (CSD) suggested that the miRNA/target pairing requirements differ between the two modes of action (cleavage vs. translational repression) (Dugas and Bartel, 2008). Note that the mechanisms of translational inhibition are still highly controversial, and proposed mechanisms include inhibition of translation initiation, inhibition of translation elongation, post-translational or co-translational protein degradation and sequestration of mRNA targets in subcellular structures, such as P bodies or stress granules (reviewed in Valencia-Sanchez et al., 2006).
A major biological function of miRNAs is in controlling development. Many plant miRNAs target transcription factor genes (Rhoades et al., 2002), which tend to play important roles in developmental regulation. In fact, a number of miRNAs, such as miR172, miR319 and miR164, were discovered in genetic screens because either their loss-of-function or over-expression led to developmental defects (Aukerman and Sakai, 2003; Palatnik et al., 2003; Baker et al., 2005). Some miRNA-target modules are conserved within or beyond angiosperms. For example, miR165/166 helps restrict the homeodomain leucine zipper genes PHBULOSA, PHAVOLUTA and REVOLUTA, which specify adaxial identity in leaves, to the adaxial domain in both Arabidopsis and maize (Zea mays) (McConnell et al., 2001; Emery et al., 2003; Juarez et al., 2004; Mallory et al., 2004). Like many miRNA-target regulatory modules, the regulation of AP2-domain protein genes by miR172 is conserved in diverse plant species. However, the biological functions of the regulatory modules may vary in different species. In Arabidopsis, miR172 promotes flowering and the determinacy of floral meristems (Aukerman and Sakai, 2003; Chen, 2004). In maize, miR172 regulates sex determination and meristem activities, and is also implicated in vegetative phase transition (Lauter et al., 2005; Chuck et al., 2007). In potato (Solanum tuberosum), miR172 promotes flowering and induces tuber formation (Martin et al., 2009). It is interesting to note that in species as divergent as Arabidopsis and C. elegans, miRNAs are employed as regulators of developmental transitions. In C. elegans, the first two miRNAs discovered, lin-4 and let-7, promote the transition from L1 to L2 larval stages, and from the L4 larval stage to adulthood, respectively (Lee et al., 1993; Reinhart et al., 2000). In Arabidopsis, a gradual decrease in miR156 levels as the plant ages leads to a gradual increase in miR172 levels to ensure proper vegetative phase transitions and the vegetative-to-reproductive transition (Wang et al., 2009; Wu et al., 2009).