- Email: [email protected]
Missing Pieces in the Puzzle of Plant MicroRNAs
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Opinion
Missing Pieces in the Puzzle of Plant MicroRNAs Rodrigo S. Reis,1,2,* Andrew L. Eamens,3 and Peter M. Waterhouse4 Plant microRNAs (miRNAs) are important regulatory switches. Recent advances have revealed many regulatory layers between the two essential processes, miRNA biogenesis and function. However, how these multilayered regulatory processes ultimately control miRNA gene regulation and connects miRNAs and plant responses with the surrounding environment is still largely unknown. In this opinion article, we propose that the miRNA pathway is highly dynamic and plastic. The apparent flexibility of the miRNA pathway in plants appears to be controlled by a number recently identified proteins and poorly characterized signaling cascades. We further propose that altered miRNA accumulation can be a direct consequence of the rewiring of interactions between proteins that function in the miRNA pathway, an avenue that remains largely unexplored. Small Noncoding RNAs in Plants
Trends Plant miRNAs are produced in nuclear dicing bodies (D-bodies). Plant miRNAs can guide either transcript cleavage or translation inhibition, and these mechanisms of silencing are defined by the dicer partnering proteins, presumably in the D-bodies. Newly discovered proteins involved in D-body formation and activity suggest the presence of a complex network of connections among D-body activity, signaling cascades, and responses to the surrounding environment.
In eukaryotic RNA silencing, double-stranded RNA (dsRNA) triggers are processed into small noncoding regulatory RNAs (sRNAs) by a member of the Dicer (see Glossary) family of endonucleases [1]. sRNAs are then loaded onto a member of the ARGONAUTE (AGO) protein family to form the catalytic core of the RNA-induced silencing complex (RISC) [2]. Endogenous or exogenous transcripts carrying complementary sequences to the RISC-loaded sRNAs are targeted by RISC to repress target gene expression at either the transcriptional or posttranscriptional level [3]. Plant sRNAs additionally require the activity of HUA ENHANCER1 (HEN1), an sRNA-specific methyltransferase to stabilize and to distinguish the sRNA from other, possibly nonfunctional, noncoding RNAs [4]. MicroRNAs (miRNAs) are a highly conserved sRNA class in plants and animals, but only in plants does their biogenesis require the assembling of a subnuclear structure, the dicing body (D-body) [5]. The stem-loop folding structure adopted by plant primary miRNAs (pri-miRNAs) is recognized and processed into mature and passenger strand miRNAs (miRNA/miRNA*) by the D-body core proteins DICER-LIKE1 (DCL1), DOUBLE-STRANDED RNA-BINDING1 (DRB1), and SERRATE (SE) [6–8]. Following duplex strand separation, DRB1 directs the selective loading of miRNA guide strands onto AGO1 to form miRNA-loaded RISC (miRISC) [9]. In plants, miRISC can repress the expression of highly complementary target transcripts via directing either a transcript cleavage or translation inhibition, and the selection of either mechanism is determined by DCL1 partnering proteins, DRB1 (cleavage) and DRB2 (translation inhibition) [10] (Figure 1). Numerous proteins have been recently demonstrated to play roles in the plant miRNA pathway, evidencing that this pathway involves a complex network of protein–protein interactions. However, the biological consequences of rewiring such protein–protein interactions, via either differential gene expression or post-transcriptional modifications, are still largely unknown. Here, we propose that protein network rewiring, post-transcriptional modification, and signaling cascades have major influences on miRNA accumulation (Figure 2, Key Figure). [4_TD$IF]A better
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1 Faculty of Agriculture and Environment, University of Sydney, Eveleigh, NSW, Australia 2 Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland 3 School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia 4 Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, QLD 4001, Australia
*Correspondence: [email protected] (R.S. Reis)
http://dx.doi.org/10.1016/j.tplants.2015.08.003 © 2015 Elsevier Ltd. All rights reserved.
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Figure 1. Simplified View of MicroRNA (miRNA) Biogenesis and Function. Primary miRNAs (pri-miRNAs) are [3_TD$IF] substrates of and are processed by DCL1 bound to SE and either DRB1 or DRB2. The methyltransferase HEN1 transfers a methyl group to the 30 terminus of each strand of the miRNA/miRNA* duplex to stabilize the small RNAs. (A) Following duplex strand separation, DRB1 directs the selective loading of miRNAs onto AGO1 to form miRISC, which in turn guides[1_TD$IF] transcript cleavage of complementary mRNAs. (B) DRB2 presumably has similar interactions and activity as those of DRB1; however, its involvement in the miRNA pathway guides[1_TD$IF] miRISC to repress translation of complementary mRNAs, possibly via inactivation of the cleavage activity of AGO. Abbreviations: DCL1, DICER-LIKE1; DRB1, DOUBLE-STRANDED RNABINDING1; DRB2, DOUBLE-STRANDED RNA-BINDING2; HEN1, HUA ENHANCER1; miRISC, miRNA-induced silencing complex; SE, SERRATE; AGO, ARGONAUTE.
understanding of how plants regulate such processes could be key to advancing our current knowledge on how miRNAs mediate responses to developmental cues and adaptation to the surrounding environment.
Current Understanding of miRNA Biogenesis: Still a Sea of Possibilities Although our knowledge of plant miRNA biogenesis and the mechanisms of miRNA-guided silencing have greatly improved over the past decade, recent findings suggest that some important aspects of the plant miRNA pathway remain poorly understood. The biogenesis of plant miRNAs is localized to nuclear D-bodies and requires a growing number of functionally diverse proteins (reviewed in [11]). Surprisingly, about half of the proteins required for D-body assembly and function were only recently identified, including TOUGH (TGH) [12], C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1) [13], DRB2 [14], MODIFIER OF SNC1, 2 (MOS2) [15], NEGATIVE ON TATA LESS2A (NOT2) [16], RECEPTOR FOR ACTIVATED C KINASE
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ALTERED MERISTEM PROGRAM1 (AMP1): integral ER membrane protein that is specifically required for miRNA-mediated translational inhibition but not transcript cleavage. Argonaute (AGO): defined by the presence of Piwi/Argonaute/Zwille (PAZ) and PIWI domains; the PIWI domain confers an RNaseH-like activity that cleaves sRNA (miRNA and siRNA) target transcripts. Dicer: an endoribonuclease responsible for cleaving long dsRNAs into short dsRNA duplexes of a specific length; biogenesis of small RNAs. In plants, dicers are known as DICER-LIKE (DCL) proteins. Dicing body (D-body): subnuclear foci that contains DCL1 and primiRNAs, as well as several other components for miRNA biogenesis. HUA ENHANCER1 (HEN1): methylates both miRNA/miRNA* and siRNA/siRNA* duplexes at their 30 termini, which protects them from uridylation and subsequent degradation. MicroRNAs (miRNAs): 20- to 24nucleotide single-stranded RNAs processed from a stem-loop region of a longer transcript by a dicer enzyme. MODIFIER OF SNC1, 2 (MOS2): binds to pri-miRNAs and is required for DRB1 recruitment of pri-miRNAs and its localization to D-bodies. NEGATIVE ON TATA LESS2A (NOT2): core member of the CARBON CATABOLITE REPRESSION4 (CCR4)–NOT complex that is involved in RNA metabolism. RECEPTOR FOR ACTIVATED C KINASE 1 (RACK1): interacts with the ribosomal machinery, numerous cell surface receptors, and nuclear proteins; key mediator of various pathways and contributes to numerous aspects of cellular function. RNA-induced silencing complex (RISC): the minimal RISC contains an Argonaute protein bound to a sRNA (miRISC, if sRNA is an miRNA); catalytic complex that confers function to a small RNA.
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Key Figure
Network View of the MicroRNA (miRNA) Pathway Key: Post-transcriponal modificaon
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Figure 2. The miRNA biogenesis and function of the miRNA pathway are highly connected processes by protein–protein interactions (depicted by the circles and lines). This interaction network appears to be modulated by gene expression changes and post-transcriptional modifications leading to rewiring of protein–protein interactions. That is, altered transcript of protein levels may influence the formation of particular interactions (e.g., via competition for interaction sites of partnering proteins), and post-transcriptional modification status may alter a protein's ability to interact with a particular protein. The rewiring of protein–protein interactions in the miRNA pathway is likely to regulate both miRNA production and function, independently of MIRNA gene expression. Note that no particular protein is depicted in this figure and interactions are also fictitious.
1 (RACK1) [17], SICKLE (SIC) [18], DAWDLE (DDL) [19], and STABILIZED 1 (STA1) [20]. Until the identification of this functionally diverse group of D-body protein machinery, our knowledge of miRNA biogenesis was largely restricted to the functional roles played by D-body core proteins, DCL1, DRB1, and SE [6,7]. The various functions of these newly identified genes and their involvement in the miRNA biogenesis suggest that this process is more complex and dynamic than previously thought. For instance, interaction of NOT2 with RNA polymerase II (PolII) is required for efficient transcription of miRNA genes (MIRNA loci) as well as protein-coding genes; specifically, NOT2 interacts with DCL1 and SE, thereby connecting MIRNA transcription to pri-miRNA processing [16]. In not2a2b double mutants, pri-miRNA expression, and hence mature miRNA accumulation is reduced, possibly as a consequence of mislocalization of DCL1 (but not DRB1) to D-bodies. This suggests that D-body assembly is independent of DCL1, as evidenced by wildtype subcellular localization of DRB1 in the not2a2b double mutants. In mos2 mutants, however, DRB1, but not DCL1, fails to localize to D-bodies and, surprisingly, MOS2 does not interact with DRB1, DCL1, or SE protein. DRB1 and pri-miRNA interaction is greatly reduced in mos2, and it is proposed that MOS2 acts by facilitating the recruitment of pri-miRNAs to D-bodies, and that
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pri-miRNAs might function as scaffolding transcripts during D-body formation [15]. Functional analysis of both NOT2ab and MOS2 provides evidence that, counterintuitively,[1_TD$IF] D-body assembly is independent of the miRNA core proteins alone. This raises the fundamental question of whether D-bodies are essential for pri-miRNA processing, and whether they play roles beyond miRNA biogenesis. The [5_TD$IF]identified [6_TD$IF]roles for CPL1 and DDL in[1_TD$IF] miRNA biogenesis also added another level of complexity to the miRNA pathway. Phosphorylated DRB1 is inactive and CPL1 is required for its activation via dephosphorylation, which in turn is essential for accurate and efficient miRNA processing by DCL1 and the preferential selection and RISC incorporation of the miRNA guide strand [13]. However, the enzyme that phosphorylates DRB1 leading to its inactivation remains unknown and, more importantly, the biological significance of having a pool of inactive phosphorylated DRB1 is also unclear. Hyperphosphorylated DRB1 is [7_TD$IF]readily detected, and the authors speculated that there is a substantial reservoir of inactive DRB1 with unknown function, perhaps acting during seed germination [13]. DDL is required for miRNA biogenesis via its ability to bind to RNA and to interact with phosphorylated forms of DCL1 [19,21,22]. Moreover, DDL contains a phosphothreonine-binding forkhead-associated (FHA) domain, often required for interaction with proteins involved in signal transduction in mammals [22]. SMAD signal transducer, structurally similar to Arabidopsis (Arabidopsis thaliana) DDL [22], integrates miRNA biogenesis and signal transduction in humans [23], and DDL appears to also provide such connection in plants [22]. Signal transduction is tightly linked to hormone responses and post-translational modifications (e.g., phosphorylation), but this connection has been given little attention in miRNA studies yet.
Current Understanding of miRNA Activity: More Than Cutting miRNA activity has been almost exclusively studied at the transcript level of the target genes, while translation inhibition is assumed to be less prevalent and an alternative pathway in plants [11]. However, recent advances suggest that miRNA-guided translational inhibition is a major component of miRNA activity. For example, in floral tissues, miRNA targets preferentially undergo translational inhibition, with limited transcript cleavage [10,24,25]. Furthermore, it has recently been demonstrated that the expression of artificial miRNAs (amiRNAs) predominantly mediate highly specific translational repression with limited mRNA decay or cleavage [26]. In addition, the endogenous and well-characterized miRNA, miR398, plays an important role in abiotic stress response, and regulates the expression of its target genes via translation inhibition as well as cleavage [27,28]. Furthermore, we recently showed that DRB2 has been much more conserved in evolution than DRB1, suggesting that translation inhibition (mediated by DRB2) is the ancient miRNA regulatory mechanism in plants. Although translational inhibition has been given much less attention than transcript cleavage, recent reports have revealed some genes that are required for this pathway, such as KATANIN1 (KTN1) [29], VARICOSE (VCS) [29], ALTERED MERISTEM PROGRAM1 (AMP1) [30], ‘SHUTTLE’ IN CHINESE (SUO) [31], and DRB2 [10]. KTN1 is a microtubule severing enzyme that is essential for the correct organization of cortical microtubules [32]. Therefore, the identification of KTN1 as a component of the translation inhibition pathway suggests that trafficking or assembly of cellular compartments involved in this pathway require the microtubule network. The involvement of AMP1, an integral endoplasmic reticulum (ER) enzyme [30], in translation inhibition further indicates that miRISC is transported from the nucleus to specific cytoplasmic sites possibly aided by the microtubule network. AGO1 appears to be the core enzyme in both cleavage and translation inhibition pathways [29,33], consequently its cleavage activity must be somehow suppressed for translation inhibition to take place. What determines this process is unknown, and we speculate that tissue and cellular context may determine AGO1 partnering proteins, thereby forming functionally distinct complexes that ultimately result in either slicing or translation inhibition activity. Indeed, the role of DRB2 in determining translation inhibition
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corroborates this hypothesis [10], as DRB2 may bridge an interaction between AGO1 and a modifier protein (e.g., kinase) hindering its cleavage activity (Figure 1). However, both DRB2– AGO1 and DRB2-‘modifier protein’ interactions remain to be experimentally demonstrated. Although little is known about AGO1 protein interactions in plants, the identification of RACK1–AGO1 interaction may shed light on the selection of miRNA regulatory mechanisms, that is, how AGO1 can be switched from ‘slicer’ to ‘blocker’ of transcripts. RACK1 is a conserved protein harboring [8_TD$IF]domains involved in simultaneous interactions with multiple proteins acting as a scaffolding protein [34]. Arabidopsis RACK1 was identified via a yeast two-hybrid screen using SE as bait, and rack1 mutants accumulate less miRNA [17]. RACK1 alters miRNA accumulation and activity via two distinct mechanisms: (i) it is required for pri-miRNA accumulation and processing precision, possibly via interaction with SE; and (ii) it is part of an AGO1 complex (200–400 kDa) [17]. The role of RACK1 in the AGO1 complex remains unknown, but it appears that it does not directly alter AGO1 slicer activity [17]. Moreover, in rack1 mutants, miR398 targets CSD1, CSD2, and CCS had increased protein accumulation without a corresponding elevation in transcript levels, suggesting miR398-guided translational inhibition. However, miR398 accumulation was decreased in rack1, also indicating a canonical explanation to the elevated target protein levels [17]. In addition to the complex role of RACK1 in the miRNA pathway, more recently, a protease–G-protein–RACK1–MAPK cascade module has been reported in which pathogen-secreted proteases activate a signaling pathway that requires RACK1 as[9_TD$IF] a scaffolding protein [35]. This newly discovered signaling pathway provides further evidence of the complex network assembled by RACK1, which may be an important connection between miRNAs and signaling pathways.
Network of miRNA Pathway Players: Joining the Dots Protein–protein interaction in the miRNA pathway has also been poorly explored. Although most reports of newly discovered miRNA players include details on their interactions, or lack of interaction, with core proteins (e.g., DCL1, DRB1, SE, and AGO1), little to no information exists on how such interactions differ between tissues, or in response to the changing environment. Such a systems biology approach would reveal an additional level of self-regulation that is likely to play a major role in controlling the miRNA pathway. That is, the selection of the regulatory [10_TD$IF] mechanism is defined by the availability of certain components, such as DRB2 [10], supporting the notion that dynamic rewiring of interactions controls the miRNA pathway. To shed light on these processes, we constructed a protein–protein interaction network using the currently reported players and their interactions (Figure 3). Gene expression data were used to assess the (i) steady-state average gene expression levels and to correlate these between different tissues (Figure 3A), and (ii) alterations in average gene expression upon perturbation and to correlate these under different perturbations (Figure 3B). The miRNA pathway requires components of protein complexes involved in transcription, 50 cap-binding complex, and spliceosome, in addition to miRNA pathway-specific proteins [11,36]. In steady-state conditions, genes such as DRB1, SQN, HEN1, and AMP1 are expressed at relatively low levels, while RACK1, AGO1, and VCS showed the highest expression levels (Figure 3A). Interestingly, most genes connected via protein–protein interactions showed high expression correlation across different tissue samples, suggesting that regardless of their level of expression, there is a bias towards their expression in similar tissues. Moreover, DRB1–DRB2 and DRB1–AGO1 gene expression did not correlate (P < 0.05), further supporting a previous report that DRB2 represses DRB1 transcription [10]. Perturbations such as abiotic and biotic stresses and hormone treatment altered the expression and the correlation between several protein partners. Counterintuitively, HST, SIC, and DRB1 expression was largely unaffected in over 3000 perturbations. HST is involved in nucleus-to-cytoplasm export of miRNAs [37], and our analyses suggest that plants do not modulate this process via HST
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Figure 3. MicroRNA (miRNA) Pathway Protein–Protein Interaction Network. Protein–protein interaction network delineated by binary interactions obtained from BIOGRID [41]. Proteins involved in the miRNA pathway without known interactor(s) are shown as isolated nodes. Nodes are colored according to gene expression obtained from Genevestigator [42]. (A) In steady-state conditions, average gene expression was determined using all occurrences available in the Genevestigator database (over 10 000 experiments) and are therefore reflective of steady-state expression. (B) In perturbed conditions, the ratios (number of occurrences for downregulation and upregulation, P < 0.05)/(total number of experiments under perturbed conditions; >3000) are given as indicative for perturbation degree for each miRNA pathway protein. Edges are colored according to the correlation of gene expression (Person's correlation; thin lines, not significant; thick lines, P < 0.05; thickest lines, P < 0.01) between two genes (nodes). In steady-state conditions, correlation refers to gene expression at the tissue level. In perturbed conditions, correlations refers to the expression fold change in the perturbation dataset. Positive and negative correlations are distinguished by solid and dashed lines, respectively. Asterisks highlight differences in correlation (edge) between steady-state and perturbed conditions. Genes without available gene expression data from Genevestigator are presented as blank nodes. AGO1 (At1g48410), AMP1 (At3g54720), CBP20 (At5g44200), CBP80 (At2g13540), CDC5 (At1g09770), CPL1 (At4g21670), DCL1 (At1g01040), DCP1 (At1g08370), DCP2 (At5g13570), DDL (At3g20550), DRB1 (At1g09700), DRB2 (At2g28380), HEN1 (At4g20910), HESO1 (At2g39740), HSP70 (At3g12580), HSP90 (At2g04030), HST (At3g05040), Mediator (At5g20170), MOS2 (At1g33520), NOT2a (At1g07705), NOT2b (At5g59710), PolII (At4g35800), PP5 (At2g42810), RACK1a (At1g18080), SAD2/EMA1 (At2g31660), SDN1 (At3g50100), SE (At2g27100), SIC (At4g24500), SQN (At2g15790), STA1 (At4g03430), SUO (At3g48050), TGH (At5g23080), VCS (At3g13300), XRN4 (At1g54490).
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abundance, even under stress conditions. Both DRB1 and SIC have been shown to be required for environmental stress adaptation [18,38], but our analyses indicate that perturbations have a relatively minor influence on the expression of either gene. It was also found that SQN, AMP1, and MOS2 expression is altered to a high degree under perturbations. SQN is essential for AGO1 function [39], possibly by aiding correct AGO1 folding, thus our analysis suggests that this process is highly affected by perturbations. Such post-transcriptional control of AGO1 activity indicates that AGO1 expression alone may not reflect the presence of functional AGO1 protein under perturbations. AMP1 is crucial for miRNA-guided translational inhibition [30], and the disproportional alteration of AMP1 expression, relative to other genes in the miRNA pathway, suggests high responsiveness of translation inhibition to perturbations, such as environmental stress. MOS2 is involved in efficient processing of pri-miRNAs, and although it does not interact with core proteins nor does MOS2 localize to D-bodies, it is required for DRB1 localization to D-bodies [15]. Thus, altered MOS2 expression under perturbations may reflect the environmentally induced effect on correct D-bodies assembly, rather than altering the expression of core players. Gene expression correlation between partnering proteins was largely unaffected by perturbations. However, there was a shift from negative to positive correlation between the 50 capbinding complex and NOT2 genes. NOT2 acts as[9_TD$IF] a scaffolding protein recruiting the miRNA biogenesis machinery to sites of MIR transcription [16], suggesting that certain perturbations trigger coordinated expression of NOT2 and 50 cap-binding complex components. Perturbations also lead to higher correlation between NOT2 and DCL1. DRB1 and TGH correlation, however, shifted from positive to negative correlation after perturbations, again suggesting that D-body formation is altered by perturbations. Altogether, these data further evidence that the miRNA pathway is inherently dynamic.
Outstanding Questions What are the underlying principles of D-body assembly? How can[1_TD$IF] D-body assembly be modulated and yet keep producing functional miRNAs? Are D-bodies an essential requirement for pri-miRNA processing? How do signaling cascades modulate the key players of the miRNA pathway? Is there a functional role for phosphorylated DRB1 and dephosphorylated DCL1 in the miRNA pathway (both appear currently inactive)? What is the role of translation inhibition in plants, and why does this regulatory mechanism appear to be more evolutionary conserved than mRNA cleavage? How does DRB2 hinder the ‘slicer’ activity of AGO1? Is translation inhibition a reversible process in plants or a step towards mRNA decay?
Concluding Remarks Our current understanding of miRNA biogenesis and activity has dramatically improved since the discovery of miRNAs as a class of regulatory RNAs [40]. However, important gaps in our understanding of these processes in plants hinder accurate prediction of their role and activity. Here, we presented a critical view of the miRNA pathway and an integration of newly identified players. This led us to some remaining questions (see Outstanding Questions) that, once addressed, should provide the missing pieces in the plant miRNA pathway puzzle and explain how this pathway is connected with the environment. This is clearly of interest to basic science, but the outcome should also benefit research in agronomically important plants. miRNAs are master regulators and detailed insights into their regulation by the environment (e.g., drought, salinity, nutrients, and heat) and how this regulation is translated into gene expression control could have far reaching implications for crop improvement. Acknowledgment We thank[1_TD$IF] Sabastine Ugbemuna Ugbaje for statistical support. R.S.R. thanks the Australian Postgraduate Awards scholarship for financial support.
References 1. Bernstein, E. et al. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 2. Hammond, S.M. et al. (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150 3. Zamore, P.D. et al. (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33
6. Vazquez, F. et al. (2004) The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Curr. Biol. 14, 346–351 7. Lobbes, D. et al. (2006) SERRATE: a new player on the plant microRNA scene. EMBO Rep. 7, 1052–1058
4. Yu, B. et al. (2005) Methylation as a crucial step in plant microRNA biogenesis. Science 307, 932–935
8. Finnegan, E.J. et al. (2003) Posttranscriptional gene silencing is not compromised in the Arabidopsis CARPEL FACTORY (DICERLIKE1) mutant, a homolog of Dicer-1 from Drosophila. Curr. Biol. 13, 236–240
5. Fang, Y. and Spector, D.L. (2007) Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Curr. Biol. 17, 818–823
9. Eamens, A.L. et al. (2009) The Arabidopsis thaliana doublestranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes. RNA 15, 2219–2235
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10. Reis, R.S. et al. (2015) Gene regulation by translational inhibition is determined by Dicer partnering proteins. Nat. Plants 1, 14027 11. Rogers, K. and Chen, X. (2013) Biogenesis, turnover, and mode of action of plant microRNAs. Plant Cell 25, 2383–2399 12. Ren, G. et al. (2012) Regulation of miRNA abundance by RNA binding protein TOUGH in Arabidopsis. Proc. Natl. Acad. Sci. U.S. A. 109, 12817–12821 13. Manavella, P.A. et al. (2012) Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1. Cell 151, 859–870 14. Eamens, A.L. et al. (2012) DRB2 is required for microRNA biogenesis in Arabidopsis thaliana. PLoS ONE 7, e35933 15. Wu, X. et al. (2013) A role for the RNA-binding protein MOS2 in microRNA maturation in Arabidopsis. Cell Res. 23, 645–657 16. Wang, L. et al. (2013) NOT2 proteins promote polymerase IIdependent transcription and interact with multiple microRNA biogenesis factors in Arabidopsis. Plant Cell 25, 715–727 17. Speth, C. et al. (2013) RACK1 scaffold proteins influence miRNA abundance in Arabidopsis. Plant J. 76, 433–445 18. Zhan, X. et al. (2012) Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis. Proc. Natl. Acad. Sci. U.S.A. 109, 18198–18203 19. Yu, B. et al. (2008) The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. Proc. Natl. Acad. Sci. U.S.A. 105, 10073–10078 20. Ben Chaabane, S. et al. (2013) STA1, an Arabidopsis pre-mRNA processing factor 6 homolog, is a new player involved in miRNA biogenesis. Nucleic Acids Res. 41, 1984–1997 21. Engelsberger, W.R. and Schulze, W.X. (2012) Nitrate and ammonium lead to distinct global dynamic phosphorylation patterns when resupplied to nitrogen-starved Arabidopsis seedlings. Plant J. 69, 978–995
26. Li, J-F. et al. (2013) Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants. Plant Cell 25, 1507–1522 27. Zhu, C. et al. (2011) MiR398 and plant stress responses. Physiol. Plant. 143, 1–9 28. Sunkar, R. et al. (2012) Functions of microRNAs in plant stress responses. Trends Plant Sci. 17, 196–203 29. Brodersen, P. et al. (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320, 1185–1190 30. Li, S. et al. (2013) MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153, 562–574 31. Yang, L. et al. (2012) Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 109, 315–320 32. Burk, D.H. et al. (2007) The katanin microtubule severing protein in plants. J. Integr. Plant Biol. 49, 1174–1182 33. Lanet, E. et al. (2009) Biochemical evidence for translational repression by Arabidopsis microRNAs. Plant Cell 21, 1762–1768 34. Adams, D.R. et al. (2011) RACK1, a multifaceted scaffolding protein: structure and function. Cell Commun. Signal. 9, 22 35. Cheng, Z. et al. (2015) Pathogen-secreted proteases activate a novel plant immune pathway. Nature 521, 213–216 36. Bologna, N.G. and Voinnet, O. (2014) The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu. Rev. Plant Biol. 65, 1–31 37. Park, M.Y. et al. (2005) Nuclear processing and export of microRNAs in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 102, 3691–3696 38. Lu, C. and Fedoroff, N. (2000) A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin. Plant Cell 12, 2351–2365
22. Machida, S. and Yuan, Y.A. (2013) Crystal structure of Arabidopsis thaliana Dawdle forkhead-associated domain reveals a conserved phospho-threonine recognition cleft for dicer-like 1 binding. Mol. Plant 6, 1290–1300
39. Smith, M.R. et al. (2009) Cyclophilin 40 is required for microRNA activity in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 106, 5424–5429
23. Davis, B.N. et al. (2008) SMAD proteins control DROSHA-mediated microRNA maturation. Nature 454, 56–61
40. Lee, R.C. and Ambros, V. (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294, 862–864
24. Chen, X. (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303, 2022–2025
41. Stark, C. et al. (2006) BioGRID: a general repository for interaction datasets. Nucleic Acids Res. 34, D535–D539
25. Grant-Downton, R. et al. (2013) Artificial microRNAs reveal cellspecific differences in small RNA activity in pollen. Curr. Biol. 23, R599–R601
42. Hruz, T. et al. (2008) Genevestigator v3: a reference expression database for the meta-analysis of transcriptomes. Adv. Bioinform. 2008, 420747
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Opinion
Missing Pieces in the Puzzle of Plant MicroRNAs Rodrigo S. Reis,1,2,* Andrew L. Eamens,3 and Peter M. Waterhouse4 Plant microRNAs (miRNAs) are important regulatory switches. Recent advances have revealed many regulatory layers between the two essential processes, miRNA biogenesis and function. However, how these multilayered regulatory processes ultimately control miRNA gene regulation and connects miRNAs and plant responses with the surrounding environment is still largely unknown. In this opinion article, we propose that the miRNA pathway is highly dynamic and plastic. The apparent flexibility of the miRNA pathway in plants appears to be controlled by a number recently identified proteins and poorly characterized signaling cascades. We further propose that altered miRNA accumulation can be a direct consequence of the rewiring of interactions between proteins that function in the miRNA pathway, an avenue that remains largely unexplored. Small Noncoding RNAs in Plants
Trends Plant miRNAs are produced in nuclear dicing bodies (D-bodies). Plant miRNAs can guide either transcript cleavage or translation inhibition, and these mechanisms of silencing are defined by the dicer partnering proteins, presumably in the D-bodies. Newly discovered proteins involved in D-body formation and activity suggest the presence of a complex network of connections among D-body activity, signaling cascades, and responses to the surrounding environment.
In eukaryotic RNA silencing, double-stranded RNA (dsRNA) triggers are processed into small noncoding regulatory RNAs (sRNAs) by a member of the Dicer (see Glossary) family of endonucleases [1]. sRNAs are then loaded onto a member of the ARGONAUTE (AGO) protein family to form the catalytic core of the RNA-induced silencing complex (RISC) [2]. Endogenous or exogenous transcripts carrying complementary sequences to the RISC-loaded sRNAs are targeted by RISC to repress target gene expression at either the transcriptional or posttranscriptional level [3]. Plant sRNAs additionally require the activity of HUA ENHANCER1 (HEN1), an sRNA-specific methyltransferase to stabilize and to distinguish the sRNA from other, possibly nonfunctional, noncoding RNAs [4]. MicroRNAs (miRNAs) are a highly conserved sRNA class in plants and animals, but only in plants does their biogenesis require the assembling of a subnuclear structure, the dicing body (D-body) [5]. The stem-loop folding structure adopted by plant primary miRNAs (pri-miRNAs) is recognized and processed into mature and passenger strand miRNAs (miRNA/miRNA*) by the D-body core proteins DICER-LIKE1 (DCL1), DOUBLE-STRANDED RNA-BINDING1 (DRB1), and SERRATE (SE) [6–8]. Following duplex strand separation, DRB1 directs the selective loading of miRNA guide strands onto AGO1 to form miRNA-loaded RISC (miRISC) [9]. In plants, miRISC can repress the expression of highly complementary target transcripts via directing either a transcript cleavage or translation inhibition, and the selection of either mechanism is determined by DCL1 partnering proteins, DRB1 (cleavage) and DRB2 (translation inhibition) [10] (Figure 1). Numerous proteins have been recently demonstrated to play roles in the plant miRNA pathway, evidencing that this pathway involves a complex network of protein–protein interactions. However, the biological consequences of rewiring such protein–protein interactions, via either differential gene expression or post-transcriptional modifications, are still largely unknown. Here, we propose that protein network rewiring, post-transcriptional modification, and signaling cascades have major influences on miRNA accumulation (Figure 2, Key Figure). [4_TD$IF]A better
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1 Faculty of Agriculture and Environment, University of Sydney, Eveleigh, NSW, Australia 2 Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland 3 School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia 4 Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, QLD 4001, Australia
*Correspondence: [email protected] (R.S. Reis)
http://dx.doi.org/10.1016/j.tplants.2015.08.003 © 2015 Elsevier Ltd. All rights reserved.
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Figure 1. Simplified View of MicroRNA (miRNA) Biogenesis and Function. Primary miRNAs (pri-miRNAs) are [3_TD$IF] substrates of and are processed by DCL1 bound to SE and either DRB1 or DRB2. The methyltransferase HEN1 transfers a methyl group to the 30 terminus of each strand of the miRNA/miRNA* duplex to stabilize the small RNAs. (A) Following duplex strand separation, DRB1 directs the selective loading of miRNAs onto AGO1 to form miRISC, which in turn guides[1_TD$IF] transcript cleavage of complementary mRNAs. (B) DRB2 presumably has similar interactions and activity as those of DRB1; however, its involvement in the miRNA pathway guides[1_TD$IF] miRISC to repress translation of complementary mRNAs, possibly via inactivation of the cleavage activity of AGO. Abbreviations: DCL1, DICER-LIKE1; DRB1, DOUBLE-STRANDED RNABINDING1; DRB2, DOUBLE-STRANDED RNA-BINDING2; HEN1, HUA ENHANCER1; miRISC, miRNA-induced silencing complex; SE, SERRATE; AGO, ARGONAUTE.
understanding of how plants regulate such processes could be key to advancing our current knowledge on how miRNAs mediate responses to developmental cues and adaptation to the surrounding environment.
Current Understanding of miRNA Biogenesis: Still a Sea of Possibilities Although our knowledge of plant miRNA biogenesis and the mechanisms of miRNA-guided silencing have greatly improved over the past decade, recent findings suggest that some important aspects of the plant miRNA pathway remain poorly understood. The biogenesis of plant miRNAs is localized to nuclear D-bodies and requires a growing number of functionally diverse proteins (reviewed in [11]). Surprisingly, about half of the proteins required for D-body assembly and function were only recently identified, including TOUGH (TGH) [12], C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1) [13], DRB2 [14], MODIFIER OF SNC1, 2 (MOS2) [15], NEGATIVE ON TATA LESS2A (NOT2) [16], RECEPTOR FOR ACTIVATED C KINASE
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ALTERED MERISTEM PROGRAM1 (AMP1): integral ER membrane protein that is specifically required for miRNA-mediated translational inhibition but not transcript cleavage. Argonaute (AGO): defined by the presence of Piwi/Argonaute/Zwille (PAZ) and PIWI domains; the PIWI domain confers an RNaseH-like activity that cleaves sRNA (miRNA and siRNA) target transcripts. Dicer: an endoribonuclease responsible for cleaving long dsRNAs into short dsRNA duplexes of a specific length; biogenesis of small RNAs. In plants, dicers are known as DICER-LIKE (DCL) proteins. Dicing body (D-body): subnuclear foci that contains DCL1 and primiRNAs, as well as several other components for miRNA biogenesis. HUA ENHANCER1 (HEN1): methylates both miRNA/miRNA* and siRNA/siRNA* duplexes at their 30 termini, which protects them from uridylation and subsequent degradation. MicroRNAs (miRNAs): 20- to 24nucleotide single-stranded RNAs processed from a stem-loop region of a longer transcript by a dicer enzyme. MODIFIER OF SNC1, 2 (MOS2): binds to pri-miRNAs and is required for DRB1 recruitment of pri-miRNAs and its localization to D-bodies. NEGATIVE ON TATA LESS2A (NOT2): core member of the CARBON CATABOLITE REPRESSION4 (CCR4)–NOT complex that is involved in RNA metabolism. RECEPTOR FOR ACTIVATED C KINASE 1 (RACK1): interacts with the ribosomal machinery, numerous cell surface receptors, and nuclear proteins; key mediator of various pathways and contributes to numerous aspects of cellular function. RNA-induced silencing complex (RISC): the minimal RISC contains an Argonaute protein bound to a sRNA (miRISC, if sRNA is an miRNA); catalytic complex that confers function to a small RNA.
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Key Figure
Network View of the MicroRNA (miRNA) Pathway Key: Post-transcriponal modificaon
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Figure 2. The miRNA biogenesis and function of the miRNA pathway are highly connected processes by protein–protein interactions (depicted by the circles and lines). This interaction network appears to be modulated by gene expression changes and post-transcriptional modifications leading to rewiring of protein–protein interactions. That is, altered transcript of protein levels may influence the formation of particular interactions (e.g., via competition for interaction sites of partnering proteins), and post-transcriptional modification status may alter a protein's ability to interact with a particular protein. The rewiring of protein–protein interactions in the miRNA pathway is likely to regulate both miRNA production and function, independently of MIRNA gene expression. Note that no particular protein is depicted in this figure and interactions are also fictitious.
1 (RACK1) [17], SICKLE (SIC) [18], DAWDLE (DDL) [19], and STABILIZED 1 (STA1) [20]. Until the identification of this functionally diverse group of D-body protein machinery, our knowledge of miRNA biogenesis was largely restricted to the functional roles played by D-body core proteins, DCL1, DRB1, and SE [6,7]. The various functions of these newly identified genes and their involvement in the miRNA biogenesis suggest that this process is more complex and dynamic than previously thought. For instance, interaction of NOT2 with RNA polymerase II (PolII) is required for efficient transcription of miRNA genes (MIRNA loci) as well as protein-coding genes; specifically, NOT2 interacts with DCL1 and SE, thereby connecting MIRNA transcription to pri-miRNA processing [16]. In not2a2b double mutants, pri-miRNA expression, and hence mature miRNA accumulation is reduced, possibly as a consequence of mislocalization of DCL1 (but not DRB1) to D-bodies. This suggests that D-body assembly is independent of DCL1, as evidenced by wildtype subcellular localization of DRB1 in the not2a2b double mutants. In mos2 mutants, however, DRB1, but not DCL1, fails to localize to D-bodies and, surprisingly, MOS2 does not interact with DRB1, DCL1, or SE protein. DRB1 and pri-miRNA interaction is greatly reduced in mos2, and it is proposed that MOS2 acts by facilitating the recruitment of pri-miRNAs to D-bodies, and that
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pri-miRNAs might function as scaffolding transcripts during D-body formation [15]. Functional analysis of both NOT2ab and MOS2 provides evidence that, counterintuitively,[1_TD$IF] D-body assembly is independent of the miRNA core proteins alone. This raises the fundamental question of whether D-bodies are essential for pri-miRNA processing, and whether they play roles beyond miRNA biogenesis. The [5_TD$IF]identified [6_TD$IF]roles for CPL1 and DDL in[1_TD$IF] miRNA biogenesis also added another level of complexity to the miRNA pathway. Phosphorylated DRB1 is inactive and CPL1 is required for its activation via dephosphorylation, which in turn is essential for accurate and efficient miRNA processing by DCL1 and the preferential selection and RISC incorporation of the miRNA guide strand [13]. However, the enzyme that phosphorylates DRB1 leading to its inactivation remains unknown and, more importantly, the biological significance of having a pool of inactive phosphorylated DRB1 is also unclear. Hyperphosphorylated DRB1 is [7_TD$IF]readily detected, and the authors speculated that there is a substantial reservoir of inactive DRB1 with unknown function, perhaps acting during seed germination [13]. DDL is required for miRNA biogenesis via its ability to bind to RNA and to interact with phosphorylated forms of DCL1 [19,21,22]. Moreover, DDL contains a phosphothreonine-binding forkhead-associated (FHA) domain, often required for interaction with proteins involved in signal transduction in mammals [22]. SMAD signal transducer, structurally similar to Arabidopsis (Arabidopsis thaliana) DDL [22], integrates miRNA biogenesis and signal transduction in humans [23], and DDL appears to also provide such connection in plants [22]. Signal transduction is tightly linked to hormone responses and post-translational modifications (e.g., phosphorylation), but this connection has been given little attention in miRNA studies yet.
Current Understanding of miRNA Activity: More Than Cutting miRNA activity has been almost exclusively studied at the transcript level of the target genes, while translation inhibition is assumed to be less prevalent and an alternative pathway in plants [11]. However, recent advances suggest that miRNA-guided translational inhibition is a major component of miRNA activity. For example, in floral tissues, miRNA targets preferentially undergo translational inhibition, with limited transcript cleavage [10,24,25]. Furthermore, it has recently been demonstrated that the expression of artificial miRNAs (amiRNAs) predominantly mediate highly specific translational repression with limited mRNA decay or cleavage [26]. In addition, the endogenous and well-characterized miRNA, miR398, plays an important role in abiotic stress response, and regulates the expression of its target genes via translation inhibition as well as cleavage [27,28]. Furthermore, we recently showed that DRB2 has been much more conserved in evolution than DRB1, suggesting that translation inhibition (mediated by DRB2) is the ancient miRNA regulatory mechanism in plants. Although translational inhibition has been given much less attention than transcript cleavage, recent reports have revealed some genes that are required for this pathway, such as KATANIN1 (KTN1) [29], VARICOSE (VCS) [29], ALTERED MERISTEM PROGRAM1 (AMP1) [30], ‘SHUTTLE’ IN CHINESE (SUO) [31], and DRB2 [10]. KTN1 is a microtubule severing enzyme that is essential for the correct organization of cortical microtubules [32]. Therefore, the identification of KTN1 as a component of the translation inhibition pathway suggests that trafficking or assembly of cellular compartments involved in this pathway require the microtubule network. The involvement of AMP1, an integral endoplasmic reticulum (ER) enzyme [30], in translation inhibition further indicates that miRISC is transported from the nucleus to specific cytoplasmic sites possibly aided by the microtubule network. AGO1 appears to be the core enzyme in both cleavage and translation inhibition pathways [29,33], consequently its cleavage activity must be somehow suppressed for translation inhibition to take place. What determines this process is unknown, and we speculate that tissue and cellular context may determine AGO1 partnering proteins, thereby forming functionally distinct complexes that ultimately result in either slicing or translation inhibition activity. Indeed, the role of DRB2 in determining translation inhibition
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corroborates this hypothesis [10], as DRB2 may bridge an interaction between AGO1 and a modifier protein (e.g., kinase) hindering its cleavage activity (Figure 1). However, both DRB2– AGO1 and DRB2-‘modifier protein’ interactions remain to be experimentally demonstrated. Although little is known about AGO1 protein interactions in plants, the identification of RACK1–AGO1 interaction may shed light on the selection of miRNA regulatory mechanisms, that is, how AGO1 can be switched from ‘slicer’ to ‘blocker’ of transcripts. RACK1 is a conserved protein harboring [8_TD$IF]domains involved in simultaneous interactions with multiple proteins acting as a scaffolding protein [34]. Arabidopsis RACK1 was identified via a yeast two-hybrid screen using SE as bait, and rack1 mutants accumulate less miRNA [17]. RACK1 alters miRNA accumulation and activity via two distinct mechanisms: (i) it is required for pri-miRNA accumulation and processing precision, possibly via interaction with SE; and (ii) it is part of an AGO1 complex (200–400 kDa) [17]. The role of RACK1 in the AGO1 complex remains unknown, but it appears that it does not directly alter AGO1 slicer activity [17]. Moreover, in rack1 mutants, miR398 targets CSD1, CSD2, and CCS had increased protein accumulation without a corresponding elevation in transcript levels, suggesting miR398-guided translational inhibition. However, miR398 accumulation was decreased in rack1, also indicating a canonical explanation to the elevated target protein levels [17]. In addition to the complex role of RACK1 in the miRNA pathway, more recently, a protease–G-protein–RACK1–MAPK cascade module has been reported in which pathogen-secreted proteases activate a signaling pathway that requires RACK1 as[9_TD$IF] a scaffolding protein [35]. This newly discovered signaling pathway provides further evidence of the complex network assembled by RACK1, which may be an important connection between miRNAs and signaling pathways.
Network of miRNA Pathway Players: Joining the Dots Protein–protein interaction in the miRNA pathway has also been poorly explored. Although most reports of newly discovered miRNA players include details on their interactions, or lack of interaction, with core proteins (e.g., DCL1, DRB1, SE, and AGO1), little to no information exists on how such interactions differ between tissues, or in response to the changing environment. Such a systems biology approach would reveal an additional level of self-regulation that is likely to play a major role in controlling the miRNA pathway. That is, the selection of the regulatory [10_TD$IF] mechanism is defined by the availability of certain components, such as DRB2 [10], supporting the notion that dynamic rewiring of interactions controls the miRNA pathway. To shed light on these processes, we constructed a protein–protein interaction network using the currently reported players and their interactions (Figure 3). Gene expression data were used to assess the (i) steady-state average gene expression levels and to correlate these between different tissues (Figure 3A), and (ii) alterations in average gene expression upon perturbation and to correlate these under different perturbations (Figure 3B). The miRNA pathway requires components of protein complexes involved in transcription, 50 cap-binding complex, and spliceosome, in addition to miRNA pathway-specific proteins [11,36]. In steady-state conditions, genes such as DRB1, SQN, HEN1, and AMP1 are expressed at relatively low levels, while RACK1, AGO1, and VCS showed the highest expression levels (Figure 3A). Interestingly, most genes connected via protein–protein interactions showed high expression correlation across different tissue samples, suggesting that regardless of their level of expression, there is a bias towards their expression in similar tissues. Moreover, DRB1–DRB2 and DRB1–AGO1 gene expression did not correlate (P < 0.05), further supporting a previous report that DRB2 represses DRB1 transcription [10]. Perturbations such as abiotic and biotic stresses and hormone treatment altered the expression and the correlation between several protein partners. Counterintuitively, HST, SIC, and DRB1 expression was largely unaffected in over 3000 perturbations. HST is involved in nucleus-to-cytoplasm export of miRNAs [37], and our analyses suggest that plants do not modulate this process via HST
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Figure 3. MicroRNA (miRNA) Pathway Protein–Protein Interaction Network. Protein–protein interaction network delineated by binary interactions obtained from BIOGRID [41]. Proteins involved in the miRNA pathway without known interactor(s) are shown as isolated nodes. Nodes are colored according to gene expression obtained from Genevestigator [42]. (A) In steady-state conditions, average gene expression was determined using all occurrences available in the Genevestigator database (over 10 000 experiments) and are therefore reflective of steady-state expression. (B) In perturbed conditions, the ratios (number of occurrences for downregulation and upregulation, P < 0.05)/(total number of experiments under perturbed conditions; >3000) are given as indicative for perturbation degree for each miRNA pathway protein. Edges are colored according to the correlation of gene expression (Person's correlation; thin lines, not significant; thick lines, P < 0.05; thickest lines, P < 0.01) between two genes (nodes). In steady-state conditions, correlation refers to gene expression at the tissue level. In perturbed conditions, correlations refers to the expression fold change in the perturbation dataset. Positive and negative correlations are distinguished by solid and dashed lines, respectively. Asterisks highlight differences in correlation (edge) between steady-state and perturbed conditions. Genes without available gene expression data from Genevestigator are presented as blank nodes. AGO1 (At1g48410), AMP1 (At3g54720), CBP20 (At5g44200), CBP80 (At2g13540), CDC5 (At1g09770), CPL1 (At4g21670), DCL1 (At1g01040), DCP1 (At1g08370), DCP2 (At5g13570), DDL (At3g20550), DRB1 (At1g09700), DRB2 (At2g28380), HEN1 (At4g20910), HESO1 (At2g39740), HSP70 (At3g12580), HSP90 (At2g04030), HST (At3g05040), Mediator (At5g20170), MOS2 (At1g33520), NOT2a (At1g07705), NOT2b (At5g59710), PolII (At4g35800), PP5 (At2g42810), RACK1a (At1g18080), SAD2/EMA1 (At2g31660), SDN1 (At3g50100), SE (At2g27100), SIC (At4g24500), SQN (At2g15790), STA1 (At4g03430), SUO (At3g48050), TGH (At5g23080), VCS (At3g13300), XRN4 (At1g54490).
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abundance, even under stress conditions. Both DRB1 and SIC have been shown to be required for environmental stress adaptation [18,38], but our analyses indicate that perturbations have a relatively minor influence on the expression of either gene. It was also found that SQN, AMP1, and MOS2 expression is altered to a high degree under perturbations. SQN is essential for AGO1 function [39], possibly by aiding correct AGO1 folding, thus our analysis suggests that this process is highly affected by perturbations. Such post-transcriptional control of AGO1 activity indicates that AGO1 expression alone may not reflect the presence of functional AGO1 protein under perturbations. AMP1 is crucial for miRNA-guided translational inhibition [30], and the disproportional alteration of AMP1 expression, relative to other genes in the miRNA pathway, suggests high responsiveness of translation inhibition to perturbations, such as environmental stress. MOS2 is involved in efficient processing of pri-miRNAs, and although it does not interact with core proteins nor does MOS2 localize to D-bodies, it is required for DRB1 localization to D-bodies [15]. Thus, altered MOS2 expression under perturbations may reflect the environmentally induced effect on correct D-bodies assembly, rather than altering the expression of core players. Gene expression correlation between partnering proteins was largely unaffected by perturbations. However, there was a shift from negative to positive correlation between the 50 capbinding complex and NOT2 genes. NOT2 acts as[9_TD$IF] a scaffolding protein recruiting the miRNA biogenesis machinery to sites of MIR transcription [16], suggesting that certain perturbations trigger coordinated expression of NOT2 and 50 cap-binding complex components. Perturbations also lead to higher correlation between NOT2 and DCL1. DRB1 and TGH correlation, however, shifted from positive to negative correlation after perturbations, again suggesting that D-body formation is altered by perturbations. Altogether, these data further evidence that the miRNA pathway is inherently dynamic.
Outstanding Questions What are the underlying principles of D-body assembly? How can[1_TD$IF] D-body assembly be modulated and yet keep producing functional miRNAs? Are D-bodies an essential requirement for pri-miRNA processing? How do signaling cascades modulate the key players of the miRNA pathway? Is there a functional role for phosphorylated DRB1 and dephosphorylated DCL1 in the miRNA pathway (both appear currently inactive)? What is the role of translation inhibition in plants, and why does this regulatory mechanism appear to be more evolutionary conserved than mRNA cleavage? How does DRB2 hinder the ‘slicer’ activity of AGO1? Is translation inhibition a reversible process in plants or a step towards mRNA decay?
Concluding Remarks Our current understanding of miRNA biogenesis and activity has dramatically improved since the discovery of miRNAs as a class of regulatory RNAs [40]. However, important gaps in our understanding of these processes in plants hinder accurate prediction of their role and activity. Here, we presented a critical view of the miRNA pathway and an integration of newly identified players. This led us to some remaining questions (see Outstanding Questions) that, once addressed, should provide the missing pieces in the plant miRNA pathway puzzle and explain how this pathway is connected with the environment. This is clearly of interest to basic science, but the outcome should also benefit research in agronomically important plants. miRNAs are master regulators and detailed insights into their regulation by the environment (e.g., drought, salinity, nutrients, and heat) and how this regulation is translated into gene expression control could have far reaching implications for crop improvement. Acknowledgment We thank[1_TD$IF] Sabastine Ugbemuna Ugbaje for statistical support. R.S.R. thanks the Australian Postgraduate Awards scholarship for financial support.
References 1. Bernstein, E. et al. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 2. Hammond, S.M. et al. (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150 3. Zamore, P.D. et al. (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33
6. Vazquez, F. et al. (2004) The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Curr. Biol. 14, 346–351 7. Lobbes, D. et al. (2006) SERRATE: a new player on the plant microRNA scene. EMBO Rep. 7, 1052–1058
4. Yu, B. et al. (2005) Methylation as a crucial step in plant microRNA biogenesis. Science 307, 932–935
8. Finnegan, E.J. et al. (2003) Posttranscriptional gene silencing is not compromised in the Arabidopsis CARPEL FACTORY (DICERLIKE1) mutant, a homolog of Dicer-1 from Drosophila. Curr. Biol. 13, 236–240
5. Fang, Y. and Spector, D.L. (2007) Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Curr. Biol. 17, 818–823
9. Eamens, A.L. et al. (2009) The Arabidopsis thaliana doublestranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes. RNA 15, 2219–2235
Trends in Plant Science, September 2015, Vol xx. No. x
7
TRPLSC 1331 No. of Pages 8
10. Reis, R.S. et al. (2015) Gene regulation by translational inhibition is determined by Dicer partnering proteins. Nat. Plants 1, 14027 11. Rogers, K. and Chen, X. (2013) Biogenesis, turnover, and mode of action of plant microRNAs. Plant Cell 25, 2383–2399 12. Ren, G. et al. (2012) Regulation of miRNA abundance by RNA binding protein TOUGH in Arabidopsis. Proc. Natl. Acad. Sci. U.S. A. 109, 12817–12821 13. Manavella, P.A. et al. (2012) Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1. Cell 151, 859–870 14. Eamens, A.L. et al. (2012) DRB2 is required for microRNA biogenesis in Arabidopsis thaliana. PLoS ONE 7, e35933 15. Wu, X. et al. (2013) A role for the RNA-binding protein MOS2 in microRNA maturation in Arabidopsis. Cell Res. 23, 645–657 16. Wang, L. et al. (2013) NOT2 proteins promote polymerase IIdependent transcription and interact with multiple microRNA biogenesis factors in Arabidopsis. Plant Cell 25, 715–727 17. Speth, C. et al. (2013) RACK1 scaffold proteins influence miRNA abundance in Arabidopsis. Plant J. 76, 433–445 18. Zhan, X. et al. (2012) Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis. Proc. Natl. Acad. Sci. U.S.A. 109, 18198–18203 19. Yu, B. et al. (2008) The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. Proc. Natl. Acad. Sci. U.S.A. 105, 10073–10078 20. Ben Chaabane, S. et al. (2013) STA1, an Arabidopsis pre-mRNA processing factor 6 homolog, is a new player involved in miRNA biogenesis. Nucleic Acids Res. 41, 1984–1997 21. Engelsberger, W.R. and Schulze, W.X. (2012) Nitrate and ammonium lead to distinct global dynamic phosphorylation patterns when resupplied to nitrogen-starved Arabidopsis seedlings. Plant J. 69, 978–995
26. Li, J-F. et al. (2013) Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants. Plant Cell 25, 1507–1522 27. Zhu, C. et al. (2011) MiR398 and plant stress responses. Physiol. Plant. 143, 1–9 28. Sunkar, R. et al. (2012) Functions of microRNAs in plant stress responses. Trends Plant Sci. 17, 196–203 29. Brodersen, P. et al. (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320, 1185–1190 30. Li, S. et al. (2013) MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153, 562–574 31. Yang, L. et al. (2012) Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 109, 315–320 32. Burk, D.H. et al. (2007) The katanin microtubule severing protein in plants. J. Integr. Plant Biol. 49, 1174–1182 33. Lanet, E. et al. (2009) Biochemical evidence for translational repression by Arabidopsis microRNAs. Plant Cell 21, 1762–1768 34. Adams, D.R. et al. (2011) RACK1, a multifaceted scaffolding protein: structure and function. Cell Commun. Signal. 9, 22 35. Cheng, Z. et al. (2015) Pathogen-secreted proteases activate a novel plant immune pathway. Nature 521, 213–216 36. Bologna, N.G. and Voinnet, O. (2014) The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu. Rev. Plant Biol. 65, 1–31 37. Park, M.Y. et al. (2005) Nuclear processing and export of microRNAs in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 102, 3691–3696 38. Lu, C. and Fedoroff, N. (2000) A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin. Plant Cell 12, 2351–2365
22. Machida, S. and Yuan, Y.A. (2013) Crystal structure of Arabidopsis thaliana Dawdle forkhead-associated domain reveals a conserved phospho-threonine recognition cleft for dicer-like 1 binding. Mol. Plant 6, 1290–1300
39. Smith, M.R. et al. (2009) Cyclophilin 40 is required for microRNA activity in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 106, 5424–5429
23. Davis, B.N. et al. (2008) SMAD proteins control DROSHA-mediated microRNA maturation. Nature 454, 56–61
40. Lee, R.C. and Ambros, V. (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294, 862–864
24. Chen, X. (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303, 2022–2025
41. Stark, C. et al. (2006) BioGRID: a general repository for interaction datasets. Nucleic Acids Res. 34, D535–D539
25. Grant-Downton, R. et al. (2013) Artificial microRNAs reveal cellspecific differences in small RNA activity in pollen. Curr. Biol. 23, R599–R601
42. Hruz, T. et al. (2008) Genevestigator v3: a reference expression database for the meta-analysis of transcriptomes. Adv. Bioinform. 2008, 420747
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