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B RIEFINGS IN FUNC TIONAL GENOMICS . VOL 12. NO 1. 37^ 45 doi:10.1093/bfgp/els050 Processing of plant microRNA precursors Nicola¤s G. Bologna, Arnaldo L. Schapire and Javier F. Palatnik Advance Access publication date 11 November 2012 Abstract MicroRNAs are endogenous small RNAs known to be key regulators of gene expression in animals and plants. They are defined by their specific biogenesis which involves the precise excision from an imperfect fold-back precursor. These precursors contain structural determinants required for their correct processing. Still, there are significant differences in the biogenesis and activities of plant and animal microRNAs. This review summarizes diverse aspects of precursor processing in plants, contrasting them to their animal counterparts. Keywords: microRNA; precursor; structural determinants; biogenesis; processing; Arabidopsis thaliana; plants; animals INTRODUCTION Small RNAs (sRNAs) are major regulators of gene expression in animals and plants. They are usually 19–24 nt long and processed from double-stranded RNAs (dsRNAs) by Rnase-III enzymes [1, 2]. Plant sRNAs can be classified into different groups such as microRNAs (miRNAs), small interfering RNAs (siRNAs), trans-acting siRNAs, natural antisensesiRNAs and the recently discovered diRNAs, which are generated after DNA lesions [3–6]. Although each class possesses its own characteristics in terms of their biogenesis and size, all of them are ultimately incorporated into RNA silencing complexes, which contain a member of the ARGONAUTE (AGO) family as a main component [3]. The sRNAs guide AGO proteins to target RNAs using base complementarity as a search tool. In turn, AGO complexes regulate gene expression at transcriptional or post-transcriptional level [2]. MiRNAs are an appealing group of sRNAs, as they originate from endogenous loci and regulate other target RNAs than those generating the sRNA [2, 7]. Plant miRNAs are usually around 21–22 nt long and mediate gene silencing at post-transcriptional level [8, 9], which entails the (i) endonucleolytic cleavage (slicing) and/or (ii) translational repression of a target mRNA. The importance of miRNA-mediated regulation is evident by the severe pleiotropic phenotypes in miRNA-deEcient mutants [10–13]. The miRNA-binding sites in target RNAs usually have an extensive sequence complementarity to the miRNAs and most experimentally validated targets contain five mismatches or less [14–16]. MiRNAs that are conserved during evolution generally regulate transcription factors that control plant development [17]. Plant miRNAs regulate few targets, usually duplicated members of one protein-coding gene family, yet, disruption of this regulation causes severe defects [2]. In contrast, animal miRNAs regulate many target genes through the base pairing of their 6-nt seed region [18]. However, the biological role of this regulation is unclear in most cases. Although miRNAs mediate diverse aspects of development and physiology in both plants and animals, there are substantial differences between them, Corresponding author. Javier F. Palatnik, IBR (Instituto de Biologı́a Molecular y Celular de Rosario), CONICET and Facultad de Ciencias Bioquı́micas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina. E-mail: [email protected] Nicola¤s G. Bologna, PhD, was a graduate student at the IBR, and is currently a postdoctoral fellow at the ETH, Switzerland. His research focuses on studying microRNAs in plants. Arnaldo L. Schapire, PhD, is a postdoctoral fellow at the IBR Institute in Rosario, Argentina. His research focuses on studying the mechanisms of microRNA processing in plants. Javier F. Palatnik, PhD, is a group leader at the IBR Institute in Rosario, Argentina. His research group studies different aspects of the biology of microRNAs. ß The Author 2012. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected] 38 Bologna et al. suggesting they have appeared independently in the two lineages [19]. Still, plant and animal miRNAs are processed from precursors containing imperfect stem–loops and generally regulate longer target mRNAs in both systems. Here, we focus on the processing of plant miRNA precursors and compare them with their animal counterparts. MiRNA BIOGENESIS PATHWAY: AN OVERVIEW Most plant miRNAs are generated from their own transcriptional units. They are transcribed by RNA polymerase II and then capped, spliced and polyadenylated [20, 21]. Although many animal miRNAs are derived from introns or untranslated regions of coding messages or primary transcripts containing tandem precursors [22, 23], most plant miRNAencoding loci comprise independent, non-proteincoding transcription units [24]. However, there are some known examples of transcripts harboring tandem precursors in plants [25, 26] and precursors located in mRNA untranslated regions [27]. The miRNA primary transcripts contain an internal stem–loop secondary structure (miRNA precursor) with the miRNA located in one of the arms. The miRNA processing machinery recognizes structural determinants in the miRNA precursors and produces staggered cleavages in the dsRNA, separated 21 nt of each other. These cuts release the miRNA together with the opposing fragment of the precursor that is interacting with it, called miRNA* (Figure 1A). The core component of the miRNA processing machinery in Arabidopsis is DICER-LIKE1 (DCL1), which is the RNAse type III that produces all cuts in the miRNA precursors [10, 11, 28]. DCL1 is assisted by the dsRNA-binding protein HYPONASTIC LEAVES1 (HYL1) [29, 30] and the C2H2 zinc-finger protein SERRATE (SE) [31, 32] (Figure 1). Both HYL1 and SE, improve the efficiency and precision of cleavage by DCL1 [33]. Although not lethal, hyl1 null mutations severely impair miRNA maturation, which in turn causes developmental defects [30, 34, 35]. Mutations in SE accumulate high levels of miRNA primary transcripts and less mature miRNAs [31, 32]. These mutants exhibit also general mRNA splicing defects [36]. ABH1/CBP80 and CBP20, which encode subunits of the nuclear CBC also display general mRNA splicing defects and accumulate more miRNA primary transcripts [36, 37]. Thus, dual roles in splicing and miRNA processing distinguish SE and CBC from HYL1, which is more specialized in miRNA biogenesis. DCL1 and HYL1 co-localize in subnuclear regions that have been termed dicing bodies or D-bodies [38–40]. Analysis of miRNA primary transcripts revealed that they are also recruited to these bodies, suggesting that these regions function as miRNA-processing centers in plants [38, 39]. The DCL1-/HYL1-containing regions have similar features as the Cajal bodies, which are known to participate in RNA metabolism (reviewed in [41]). However, while the DCL1-/HYL1-containing bodies contain SmD3 and SmB [39], they do not express coilin [38, 39, 42], a main marker of the Cajal bodies [43]. DAWDLE, a DCL1 interacting protein, is thought to stabilize miRNA primary transcripts until they are processed by DCL1 [44]. HASTY, a homolog of the animal Exportin5, also contributes to the levels of certain miRNAs [45]. However, whereas Exportin5 transports animal pre-miRNAs to the cytoplasm (see below), the molecular role of HASTY is unclear as all processing steps occur in the plant nucleus. Still, HASTY might be associated with other cargo, such as the miRNA/miRNA*. Recently, it has been reported that the lack of TOUGH, an RNA-binding protein, reduces the accumulation of miRNAs and siRNAs in vivo [46]. TOUGH binds to miRNA precursors in vivo and might aid in the recruitment of primary transcripts or contribute to the efficiency of the DCL1 processing complex. Finally, after the processing of the precursors, the miRNA/miRNA* duplex is released. The 30 -ends of both miRNA and miRNA* are 20 -O-methylated by the nuclear protein HEN1, preventing its degradation [47, 48] (Figure 1A). In contrast to plants, miRNA biogenesis is compartmentalized in animals. First, primary transcripts are trimmed in the nucleus to separate the stem– loop precursor from the rest of the transcript. This process is achieved by a Microprocessor complex formed by an RNase III-like enzyme termed Drosha and the dsRNA-binding protein DiGeorge syndrome critical region gene 8 (DGCR8; Pasha in Drosophila melanogaster and Caenorhabditis elegans) [49–51]. The fold-back precursors are then translocated into the cytoplasm by Exportin 5 [52, 53]. Once there, these stem–loops are cleaved by a Dicer, releasing 21-nt miRNA duplexes [54–56] (Figure 1B). Dicer acts in Processing of plant microRNA precursors 39 Figure 1: Overview of miRNA processing pathways in plants and animals. (A) Plant miRNA primary transcripts are stabilized by DAWDLE (DDL). In the D-bodies, DCL1, assisted by HYL1, SE and nuclear CBC cleaves the precursor to release the miRNA/miRNA* duplex. The sRNAs are stabilized by the addition of a methyl group (black dot) by HEN1. One strand of the duplex, the miRNA (red), is incorporated into an AGO protein complex. (B) In animals (humans), the initiation step (cropping) is mediated by the Drosha ^DiGeorge syndrome critical region gene 8 (DGCR8) complex (also known as the Microprocessor complex) that generates pre-miRNAs. Then, the pre-miRNA is recognized by the nuclear export factor Exportin 5 (EXP5) and is exported to the cytoplasm. The cytoplasmic RNase III Dicer catalyzes the second processing step to produce miRNA duplexes assisted by TRBP or PACT. One strand of the duplex is finally incorporated into an AGO protein. concert with other dsRNA-binding proteins: Loquacious, that contains three dsRNA-binding domains (dsRBDs) [57–59] in flies, and HIV trans-activator RNA-binding protein (TRBP) [60, 61] and protein activator of the interferon induced protein kinase (PACT) [62] in humans. In contrast to plants, animal miRNAs are usually not methylated. Still, methylation by HEN1 orthologs has been observed in other classes of sRNAs, such as the single-stranded piRNAs and certain siRNAs [48, 63–65]. 40 Bologna et al. Structural determinants of miRNA precursors The processing of the fold-back precursors by the RNAse III complexes causes the release of miRNA/miRNA* duplexes (Figure 1). Although the miRNA is incorporated into an AGO complex, the miRNA* is generally degraded. The precision in the position of the cuts along the precursor is of key importance, as they determine the sequence of the miRNA and therefore its target specificity. Of special importance is the selection of the position for the first cleavage reaction because the second cut is usually performed by measuring a fixed distance from the end of the precursor. The evidence accumulated so far, indicates that the secondary structure of the fold-back precursor directs the activity of the processing machinery represented by Drosha and DCL1 complexes. In some, the biogenesis of the miRNAs could proceed by other pathways. For example, a few recently evolved miRNAs have been shown to depend on DCL4 rather than DCL1 in Arabidopsis thaliana [27] and the activity of Drosha can be bypassed by the splicing machinery, generating mirtrons [66, 67], which have been also described in plants [68, 69]. The biogenesis of most animal and plant miRNAs, however, is canalized through Drosha and DCL1, respectively. Animal precursors A typical miRNA precursor in animals comprises a stem of approximately three helical turns (65 nt), a terminal loop and long single-stranded RNA (ssRNA) Fanking sequence below the fold-back structure (Figure 2A). The stem corresponds to the miRNA/miRNA* plus an additional 11 nt of a lower stem located below the miRNA. Experiments performed in vitro have shown that the terminal loop is dispensable for processing the miRNA precursors, whereas the region below the miRNA is essential [70, 71]. Furthermore, it was demonstrated that the distance from the ssRNA basal segments to miRNA/miRNA* duplex is critical for cleavage site selection [70]. The model for the biogenesis of animal miRNAs suggests that DGCR8/Pasha recognizes the precursor by anchoring at the ssRNA–dsRNA junction while interacting with the precursor stem. After this initial recognition, the processing center of Drosha might be located, performing the first cleavage 11 bp away from the ssRNA–dsRNA junction and releasing the fold-back from the rest of the Figure 2: Structural determinants in animal and plant miRNA precursors. (A) A typical miRNA precursor in animals comprises a stem of approximately three helical turns (miRNA/miRNA* duplex plus a lower stem), a terminal loop and long ssRNA Fanking sequences. (B) A plant miRNA precursor could be divided into four parts comprising a lower stem, the miRNA/ miRNA*, an upper stem and the terminal loop. Many plant miRNA precursors have a lower stem of 15 nt below the miRNA/miRNA* that is followed by a large bulge. (C) The current canonical ‘base-to-loop’ processing model in plants implicates an initial cleavage event at the base of precursor followed by a second cut produced at 21-nt distance of this first cut close to the terminal loop. The miRNA/miRNA* is indicated in bold lines. transcript [23, 70]. The second cleavage can then be performed by Dicer in the cytoplasm by recognizing the free 30 -end of the precursor stem and cutting 22 nt (approximately two helical turns) from this end [57]. Plant precursors In contrast to the defined in vitro systems used to analyze miRNA biogenesis in animals (e.g. [70, 71]), most of the studies carried out in plants used transgenic plants expressing miRNA precursors [72–74]. In many plant precursors, a single change in the lower stem of 15 nt below the miRNA is sufficient Processing of plant microRNA precursors to completely abolish its processing [72–75]. A random mutagenesis approach performed on miR172a precursor revealed that point mutations located in the lower stem significantly affected its processing, whereas mutations located in the terminal loop were largely neutral [72]. Interestingly, these unbiased mutations that affected miRNA processing identified by this approach also destabilized the precursor structure [72]. A systematic analysis of plant precursors revealed that many of them have a lower stem of 15 nt below the miRNA/miRNA* that follows the presence of a large bulge [72–74] (Figure 2B). The current model implicates the recognition of this lower stem to position the initial DCL1 cleavage event. The second cut is produced in the stem at 21 nt of this first cut. These structural determinants are similar but not identical to those identified in animal miRNA precursors (Figure 2C). Loop-to-base processing of plant precursors The animal stem–loop structures of miRNA precursors have highly uniform sizes of 65 nt, whereas in plants there is a wide range of precursors from 50 to >500 nt [76] (Figure 3, inset). This heterogeneity in the precursor structures might also reflect differences in the processing pathways. The precursors of miR159 and miR319 are among those with long fold-backs [77–79]. Detailed mutagenesis studies revealed that the processing of these precursors begin with a cleavage next to the loop [80]. DCL1 then continues to cut the precursor three more times at 20- to 22-nt intervals in a ‘loop-to-base’ direction, until the miRNA is finally released [80, 81] (Figure 3). Removing the lower stem below the miRNA/miRNA* did not affect miR319 processing. On the contrary, modifying the upper part of the precursor severely impaired the biogenesis of miR319 [80]. The processing of plant precursors by multiple DCL1 cuts has been also observed in other cases [28, 82] and so far, is a distinctive feature not present in animal precursors. In principle, the processing of miR319 and miR159 precursors might lead to the accumulation of several miRNAs. The experimental evidence so far indicates that miR319 and miR159 have clear biological roles in plant development [78, 80, 83–85]. In turn, miR319 and miR159 are highly conserved across the plant kingdom and copies of miR319 can even be found in mosses, also being 41 processed in a loop-to-base direction, indicating their ancient origin [80, 81, 86, 87]. The other sRNAs can be detected in vivo associated with AGO proteins [88]; however, their biological function remains to be elucidated. The secondary structure of these long precursors contains information regarding the final level of the different potential sRNAs [80]. It seems that the presence of large number of bulges in certain regions of the stem prevents the accumulation of those sRNAs [80]. Young miRNAs It was found that some newly evolved miRNA fold-backs in plants show complementarity with their target genes that extended beyond the miRNA/miRNA* region [24, 27, 89–91]. This observation strongly suggests that many young MIRNA genes derive from inverted duplication events of their targets, giving rise to ‘proto-miRNAs’ [24, 27, 89–91]. However, these newly formed RNA structures do not necessarily have the requirements for miRNA biogenesis as described above and some of them might be processed by different DCLs (reviewed in [2]). In this model, the dsRNA with perfect base pairing formed after a gene duplication event is initially processed by DCL3 and DCL4 to release several siRNAs (or proto-miRNAs). During evolution, the accumulation of mutations in the fold-back arms would lead to changes in the secondary structure and shortening of the hairpin stem, and finally, the processing will be channeled through the miRNA-specific DCL1. In accordance with this hypothesis, mir822, miR839 and miR869 fold-backs are processed by DCL4 rather than DCL1 [92, 93]. DCL3 has also shown to process conserved miRNAs of Arabidopsis [94], as well as certain precursors in rice to yield long-miRNAs (24 nt) [95]. For example, in Arabidopsis the activity of DCL3 causes the accumulation of a 24-nt species for certain miRNAs in addition to the main 21-nt class. This is most obvious in inflorescences, which is also the tissue where DCL3 transcripts accumulate [94]. The processing of miR1850 in rice leads to 21- and 24-nt miRNA species by the sequential action of DCL1 and DCL3, respectively. Furthermore, DCL1 and DCL3 can act in parallel on several other precursors as well [95]. It is certain that these recently evolved miRNAs contribute to the heterogeneity of plant precursors (Figure 3, inset). 42 Bologna et al. Figure 3: The proposed model for the biogenesis of miR319 and miR159. The miRNA precursor is first cleaved below the loop by DCL1. The processing then continues with three more cuts until the miRNA/miRNA* is released (indicated in bold lines). The miRNA is subsequently incorporated into an AGO complex. Inset: size distribution of the fold-backs of C. elegans and A. thaliana. The miRNAs from Arabidopsis are further classified into young and evolutionary conserved miRNAs (at least in angiosperms). The precursors (pre-miRNAs) were considered as the sequence between the miRNA and the miRNA*. PERSPECTIVES Plant miRNA precursors are much more heterogeneous than their animal counterparts. In part, this heterogeneity resides in the base-to-loop and loop-to-base processing mechanisms that generate them, as well in the multiple cuts that can be produced by DCL1 to release the miRNAs. Young miRNAs, formed by inverted duplication of protein-coding sequences, contribute to the large variation of shapes and sizes of the plant precursors. Still, a more systematic approach is needed to understand the extension of the complexity of miRNA processing in plants. Interestingly, it has been recently shown that precursors containing a structural asymmetry due to the presence of bulges in either the miRNA or the miRNA*, will then generate sRNAs capable of transitivity [96]. It has been suggested that AGO1 complexes with these sRNAs recruit RDR6 and SGS3, which in turn generate dsRNA from their targets and secondary siRNAs [96], therefore, linking the processing of the miRNAs with their activity. It would be interesting to determine the molecular mechanisms and the causal relationships between the processing of the precursor and the activity of the sRNAs. Key Points Processing of plant microRNA precursors. Comparison of animal and plant microRNA biogenesis. Structural determinants of animal and plant microRNA precursors. FUNDING Grants from the Agencia Nacional de Promoción Cientı́fica y Tecnológica (ANPCyT) and Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (CONICET) (to J.P.). Processing of plant microRNA precursors References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136:215–33. Voinnet O. Origin, biogenesis, and activity of plant microRNAs. Cell 2009;136:669–87. Chapman EJ, Carrington JC. Specialization and evolution of endogenous small RNA pathways. Nat Rev Genet 2007;8: 884–96. Ramachandran V, Chen X. Small RNA metabolism in Arabidopsis. Trends Plant Sci 2008;13:368–74. Vaucheret H. Post-transcriptional small RNA pathways in plants: mechanisms and regulations. Genes Dev 2006;20: 759–71. Wei W, Ba Z, Gao M, et al. A role for small RNAs in DNA double-strand break repair. Cell 2012;149:101–12. Meyers BC, Axtell MJ, Bartel B, etal. Criteria for annotation of plant MicroRNAs. Plant Cell 2008;20:3186–90. Baumberger N, Baulcombe DC. Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci USA 2005;102:11928–33. Qi Y, Denli AM, Hannon GJ. Biochemical specialization within Arabidopsis RNA silencing pathways. Mol Cell 2005; 19:421–8. Park W, Li J, Song R, et al. CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr Biol 2002;12: 1484–95. Reinhart BJ, Weinstein EG, Rhoades MW, et al. MicroRNAs in plants. Genes Dev 2002;16:1616–26. Jones-Rhoades MW, Bartel DP. Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol Cell 2004;14:787–99. Sunkar R, Zhu JK. Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 2004;16: 2001–19. Mallory AC, Reinhart BJ, Jones-Rhoades MW, et al. MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 50 region. EMBOJ 2004;23:3356–64. Schwab R, Palatnik JF, Riester M, et al. Specific effects of microRNAs on the plant transcriptome. Dev Cell 2005;8: 517–27. Fahlgren N, Carrington JC. miRNA target prediction in plants. Methods Mol Biol 2010;592:51–7. Rubio-Somoza I, Weigel D. MicroRNA networks and developmental plasticity in plants. Trends Plant Sci 2011;16: 258–64. Rajewsky N. microRNA target predictions in animals. Nat Genet 2006;38:S8–13. Axtell MJ, Westholm JO, Lai EC. Vive la difference: biogenesis and evolution of microRNAs in plants and animals. Genome Biol 2011;12:221. Xie Z, Allen E, Fahlgren N, et al. Expression of Arabidopsis MIRNA genes. Plant Physiol 2005;138:2145–54. Szarzynska B, Sobkowiak L, Pant BD, et al. Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs. Nucleic Acids Res 2009;37: 3083–93. 43 22. Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs. Cell 2009;136:642–55. 23. Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 2009;10:126–39. 24. Allen E, Xie Z, Gustafson AM, et al. Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana. Nat Genet 2004;36: 1282–90. 25. Chuck G, Cigan AM, Saeteurn K, et al. The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA. Nat Genet 2007;39:544–9. 26. Boualem A, Laporte P, Jovanovic M, et al. MicroRNA166 controls root and nodule development in Medicago truncatula. PlantJ 2008;54:876–87. 27. Rajagopalan R, Vaucheret H, Trejo J, et al. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 2006;20:3407–25. 28. Kurihara Y, Watanabe Y. Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc Natl Acad Sci USA 2004;101:12753–8. 29. Han MH, Goud S, Song L, et al. The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc Natl Acad Sci USA 2004;101:1093–8. 30. Vazquez F, Gasciolli V, Cre P. The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Current Biol 2004;14:346–51. 31. Lobbes D, Rallapalli G, Schmidt DD, et al. SERRATE: a new player on the plant microRNA scene. EMBO Rep 2006;7:1052–8. 32. Yang L, Liu Z, Lu F, et al. SERRATE is a novel nuclear regulator in primary microRNA processing in Arabidopsis. PlantJ 2006;47:841–50. 33. Dong Z, Han MH, Fedoroff N. The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1. Proc Natl Acad Sci USA 2008;105: 9970–5. 34. Han M-H, Goud S, Song L, et al. The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc Natl Acad Sci USA 2004;101:1093–8. 35. Lu C, Fedoroff N. A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin. Plant Cell 2000;12: 2351–66. 36. Laubinger S, Sachsenberg T, Zeller G, etal. Dual roles of the nuclear cap-binding complex and SERRATE in premRNA splicing and microRNA processing in Arabidopsis thaliana. Proc Natl Acad Sci USA 2008;105:8795–800. 37. Gregory BD, O’Malley RC, Lister R, et al. A link between RNA metabolism and silencing affecting Arabidopsis development. Dev Cell 2008;14:854–66. 38. Fang Y, Spector DL. Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Curr Biol 2007;17:818–23. 39. Fujioka Y, Utsumi M, Ohba Y, et al. Location of a possible miRNA processing site in SmD3/SmB nuclear bodies in Arabidopsis. Plant Cell Physiol 2007;48:1243–53. 40. Song L, Han M-H, Lesicka J, et al. Arabidopsis primary microRNA processing proteins HYL1 and DCL1 define a 44 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. Bologna et al. nuclear body distinct from the Cajal body. Proc Natl Acad Sci USA 2007;104:5437–42. Pontes O, Pikaard CS. siRNA and miRNA processing: new functions for Cajal bodies. Curr Opin Genet Dev 2008;18: 197–203. Song L, Han M-H, Lesicka J, et al. Arabidopsis primary microRNA processing proteins HYL1 and DCL1 define a nuclear body distinct from the Cajal body. Proc Natl Acad Sci USA 2007;104:5437–42. Collier S, Pendle A, Boudonck K, et al. A distant coilin homologue is required for the formation of cajal bodies in Arabidopsis. Mol Biol Cell 2006;17:2942–51. Yu B, Bi L, Zheng B, et al. The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. Proc Natl Acad Sci USA 2008;105: 10073–8. Park MY, Wu G, Gonzalez-Sulser A, et al. Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci USA 2005;102:3691–6. Ren G, Xie M, Dou Y, et al. Regulation of miRNA abundance by RNA binding protein TOUGH in Arabidopsis. Proc Natl Acad Sci USA 2012;109:12817–21. Boutet S, Vazquez F, Liu J, et al. Arabidopsis HEN1: a genetic link between endogenous miRNA controlling development and siRNA controlling transgene silencing and virus resistance. Curr Biol 2003;13:843–8. Yu B, Yang Z, Li J, et al. Methylation as a crucial step in plant microRNA biogenesis. Science 2005;307:932–5. Denli AM, Tops BB, Plasterk RH, et al. Processing of primary microRNAs by the Microprocessor complex. Nature 2004;432:231–5. Gregory RI, Yan KP, Amuthan G, et al. The Microprocessor complex mediates the genesis of microRNAs. Nature 2004;432:235–40. Han J, Lee Y, Yeom KH, et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev 2004;18: 3016–27. Lund E, Guttinger S, Calado A, et al. Nuclear export of microRNA precursors. Science 2004;303:95–8. Yi R, Qin Y, Macara IG, et al. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 2003;17:3011–6. Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001;409:363–6. Hutvagner G, McLachlan J, Pasquinelli AE, et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 2001;293:834–8. Ketting RF, Fischer SE, Bernstein E, etal. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 2001;15: 2654–9. Saito K, Ishizuka A, Siomi H, et al. Processing of pre-microRNAs by the Dicer-1-Loquacious complex in Drosophila cells. PLoS Biol 2005;3:e235. Jiang F, Ye X, Liu X, et al. Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila. Genes Dev 2005;19: 1674–9. Förstemann K, Tomari Y, Du T, et al. Normal microRNA maturation and germ-line stem cell maintenance requires 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol 2005;3:e236. Chendrimada TP, Gregory RI, Kumaraswamy E, et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 2005; 436:740–4. Haase AD, Jaskiewicz L, Zhang H, et al. TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep 2005;6: 961–7. Lee Y, Hur I, Park SY, etal. The role of PACT in the RNA silencing pathway. EMBO J 2006;25:522–32. Huang RH. Unique 20 -O-methylation by Hen1 in eukaryotic RNA interference and bacterial RNA repair. Biochemistry 2012;51:4087–95. Billi AC, Alessi AF, Khivansara V, et al. The Caenorhabditis elegans HEN1 ortholog, HENN-1, methylates and stabilizes select subclasses of germline small RNAs. PLoS Genet 2012;8:e1002617. Montgomery TA, Rim YS, Zhang C, et al. PIWI associated siRNAs and piRNAs specifically require the Caenorhabditis elegans HEN1 ortholog henn-1. PLoS Genet 2012;8: e1002616. Okamura K, Hagen JW, Duan H, et al. The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila. Cell 2007;130:89–100. Ruby JG, Jan CH, Bartel DP. Intronic microRNA precursors that bypass Drosha processing. Nature 2007;448:83–6. Zhu QH, Spriggs A, Matthew L, et al. A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains. Genome Res 2008;18:1456–65. Meng Y, Shao C. Large-scale identification of mirtrons in Arabidopsis and rice. PLoS One 2012;7:e31163. Han J, Lee Y, Yeom KH, et al. Molecular basis for the recognition of primary microRNAs by the DroshaDGCR8 complex. Cell 2006;125:887–901. Zeng Y, Cullen BR. Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences. J Biol Chem 2005;280:27595–603. Mateos JL, Bologna NG, Chorostecki U, etal. Identification of microRNA processing determinants by random mutagenesis of Arabidopsis MIR172a precursor. Curr Biol 2010; 20:49–54. Song L, Axtell MJ, Fedoroff NV. RNA secondary structural determinants of miRNA precursor processing in Arabidopsis. Curr Biol 2010;20:37–41. Werner S, Wollmann H, Schneeberger K, et al. Structure determinants for accurate processing of miR172a in Arabidopsis thaliana. Curr Biol 2010;20:42–8. Cuperus JT, Montgomery TA, Fahlgren N, et al. Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing. Proc Natl Acad Sci USA 2010;107:466–71. Cuperus JT, Fahlgren N, Carrington JC. Evolution and functional diversification of miRNA genes. Plant Cell 2011;23:431–42. Axtell MJ, Snyder JA, Bartel DP. Common functions for diverse small RNAs of land plants. Plant Cell 2007;19: 1750–69. Palatnik JF, Allen E, Wu X, et al. Control of leaf morphogenesis by microRNAs. Nature 2003;425:257–63. Processing of plant microRNA precursors 79. Warthmann N, Das S, Lanz C, et al. Comparative analysis of the MIR319a microRNA locus in Arabidopsis and related Brassicaceae. Mol Biol Evol 2008;25:892–902. 80. Bologna NG, Mateos JL, Bresso EG, et al. A loop-to-base processing mechanism underlies the biogenesis of plant microRNAs miR319 and miR159. EMBO J 2009;28: 3646–56. 81. Addo-Quaye C, Snyder JA, Park YB, et al. Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome. RNA 2009;15:2112–21. 82. Zhang W, Gao S, Zhou X, et al. Multiple distinct small RNAs originate from the same microRNA precursors. Genome Biol 2010;11:R81. 83. Palatnik JF, Wollmann H, Schommer C, etal. Sequence and expression differences underlie functional specialization of Arabidopsis microRNAs miR159 and miR319. Dev Cell 2007;13:115–25. 84. Schommer C, Palatnik JF, Aggarwal P, et al. Control of jasmonate biosynthesis and senescence by miR319 targets. PLoS Biol 2008;6:e230. 85. Allen RS, Li J, Stahle MI, et al. Genetic analysis reveals functional redundancy and the major target genes of the Arabidopsis miR159 family. Proc Natl Acad Sci USA 2007; 104:16371–6. 86. Arazi T, Talmor-Neiman M, Stav R, et al. Cloning and characterization of micro-RNAs from moss. Plant J 2005; 43:837–48. 87. Axtell MJ, Bartel DP. Antiquity of microRNAs and their targets in land plants. Plant Cell 2005;17:1658–73. 45 88. Sobkowiak L, Karlowski W, Jarmolowski A, et al. Non-canonical processing of Arabidopsis pri-miR319a/ b/c generates additional microRNAs to Target one RAP2.12 mRNA isoform. Front Plant Sci 2012;3:46. 89. Fahlgren N, Howell MD, Kasschau KD, et al. High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS One 2007;2:e219. 90. Fahlgren N, Jogdeo S, Kasschau KD, et al. MicroRNA gene evolution in Arabidopsis lyrata and Arabidopsis thaliana. Plant Cell 2010;22:1074–89. 91. Ma Z, Coruh C, Axtell MJ. Arabidopsis lyrata small RNAs: transient MIRNA and small interfering RNA loci within the Arabidopsis genus. Plant Cell 2010;22:1090–103. 92. Rajagopalan R, Vaucheret H, Trejo J, et al. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 2006;20:3407–25. 93. Ben Amor B, Wirth S, Merchan F, et al. Novel long non-protein coding RNAs involved in Arabidopsis differentiation and stress responses. Genome Res 2009;19:57–69. 94. Vazquez F, Blevins T, Ailhas J, et al. Evolution of Arabidopsis MIR genes generates novel microRNA classes. Nucleic Acids Res 2008;36:6429–38. 95. Wu L, Zhou H, Zhang Q, etal. DNA methylation mediated by a microRNA pathway. Mol Cell 2010;38:465–75. 96. Manavella PA, Koenig D, Weigel D. Plant secondary siRNA production determined by microRNA-duplex structure. Proc Natl Acad Sci USA 2012;109:2461–6.