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microRNA-Directed Phasing during Trans-Acting siRNA Biogenesis in Plants
Abstract
Plants and animals use small RNAs (microRNAs [miRNAs] and siRNAs) as guides for posttranscriptional and epigenetic regulation. In plants, miRNAs and trans-acting (ta) siRNAs form through distinct biogenesis pathways, although they both interact with target transcripts and guide cleavage. An integrated approach to identify targets of Arabidopsis thaliana miRNAs and ta-siRNAs revealed several new classes of small RNA-regulated genes, including conventional genes such as Argonaute2 and an E2-ubiquitin conjugating enzyme. Surprisingly, five ta-siRNA-generating transcripts were identified as targets of miR173 or miR390. Rather than functioning as negative regulators, miR173- and miR390-guided cleavage was shown to set the 21-nucleotide phase for ta-siRNA precursor processing. These data support a model in which miRNA-guided formation of a 5' or 3' terminus within pre-ta-siRNA transcripts, followed by RDR6-dependent formation of dsRNA and Dicer-like processing, yields phased ta-siRNAs that negatively regulate other genes.
... Secondary structures of each sequence were predicted using MFold (Zuker 2003). Under conditions similar to those suggested by Allen et al. (2005), a stem-loop structure with≤3 gaps involving≤8 bases at the small RNA location was considered as a candidate miRNA precursor. Sequences of newly identified miRNA precursors from our fulllength complementary (cDNA) have been submitted to GenBank under accession numbers FL488860-FL488875. ...
... Sequences complementary to the novel miRNA were then checked for existence of miRNA*. Target genes of miRNAs were predicted using the first criterion used by Allen et al. (2005), in which mismatched bases were penalized according to their location in the alignment (Allen et al. 2005). All other data processing and graphical display were performed using in-house Perl scripts. ...
... Sequences complementary to the novel miRNA were then checked for existence of miRNA*. Target genes of miRNAs were predicted using the first criterion used by Allen et al. (2005), in which mismatched bases were penalized according to their location in the alignment (Allen et al. 2005). All other data processing and graphical display were performed using in-house Perl scripts. ...
The small RNA transcriptomes of bread wheat and its emerging model Brachypodium distachyon were obtained by using deep sequencing technology. Small RNA compositions were analyzed in these two species. In addition to 70 conserved microRNAs (miRNAs) from 25 families, 23 novel wheat miRNAs were identified. For Brachypodium, 12 putative miRNAs were predicted from a limited number of expressed sequence tags, of which one was a potential novel miRNA. Also, 94 conserved miRNAs from 28 families were identified in this species. Expression validation was performed for several novel wheat miRNAs. RNA ligase-mediated 5′ rapid amplification of complementary DNA ends experiments demonstrated their capability to cleave predicted target genes including three disease-resistant gene analogs. Differential expression of miRNAs was observed between Brachypodium vegetative and reproductive tissues, suggesting their different roles at the two growth stages. Our work significantly increases the novel miRNA numbers in wheat and provides the first set of small RNAs in B. distachyon.
... Therefore, we checked whether the expression profiles of miR167, miR159, miR319c target genes exhibited On the other hand, Cai et al., (2018) described that TAS1c-siR483 and TAS2-siR453 (secondary siRNAs derived from TAS1c and TAS2 A. thaliana loci), were selectively loaded into plant extracellular vesicles, transported to the B. cinerea cells, and reduced fungal virulence by mediating cross-kingdom repression of vesicle-trafficking genes [25]. Interestingly, we found that miR173, which triggered the production of secondary siRNAs from TAS1a, TAS1b, TAS1c and TAS2 [47,48], was slightly upregulated upon B. cinerea treatment (6 hpi) (Fig 5). We also found the upregulation of secondary siRNAs derived from TAS1c and TAS2 transcripts during B. cinerea treatment (S6 and S7 Figs and Fig 5). ...
... https://doi.org/10.1371/journal.pone.0304790.g007 miR403 [40,47]. In our analysis, miR403-3p was upregulated and AGO2 was downregulated by B. cinerea infection (Fig 7). ...
In plants, small RNAs (sRNAs), mainly microRNAs (miRNAs) and small interfering RNAs (siRNAs), have been described as key regulators of plant development, growth, and abiotic and biotic responses. Despite reports indicating the involvement of certain sRNAs in regulating the interaction between Botrytis cinerea (a major necrotrophic fungal phytopathogen) and host plants, there remains a lack of analysis regarding the potential regulatory roles of plant sRNAs during early stages of the interaction despite early immune responses observed then during infection. We present the first transcriptome-wide analysis of small RNA expression on the early interaction between the necrotrophic fungus Botrytis cinerea and the model plant Arabidopsis thaliana. We found that evolutionary conserved A. thaliana miRNAs were the sRNAs that accumulated the most in the presence of B. cinerea. The upregulation of miR167, miR159 and miR319 was of particular interest because these, together with their target transcripts, are involved in the fine regulation of the plant hormone signaling pathways. We also describe that miR173, which triggers the production of secondary siRNAs from TAS1 and TAS2 loci, as well as secondary siRNAs derived from these loci, is upregulated in response to B. cinerea. Thus, at an early stage of the interaction there are transcriptional changes of sRNA-guided silencing pathway genes and of a subset of sRNAs that targeted genes from the PPR gene superfamily, and these may be important mechanisms regulating the interaction between A. thaliana and B. cinerea. This work provides the basis for a better understanding of the regulation mediated by sRNAs during early B. cinerea-plant interaction and may help in the development of more effective strategies for its control.
... 24,25 The three target genes of miR173(At2g27400, At1g50055, and At2g39675) encode tasiRNAs and are, therefore, named TAS1a, TAS1b, and TAS1c, respectively. 26,27 In particular, the TAS1c transcript was engineered to process artificially trans-acting siRNAs to confer consistent and effective gene silencing. 28,29 Replacing the endogenous siRNAs encoded in the TAS1c gene with sequences from the FAD2 gene silenced FAD2 activity to levels comparable to the fad2-1 null allele in nearly all transgenic events. ...
... In theory, tasiRNAs are thought to be derived from the DCL4cleavage of RNA precursors synthesized by RDR6. 26,52 In this case, the 21-nt syn-tasiRNAs were also processed by DCL4 in a phased manner following the miR173 cleavage site. However, the antiviral effects of the syn-tasiRNA constructs were not significant in the presence or absence of miR173. ...
Trans-acting small interfering RNAs (tasiRNAs) are 21-nt phased (phased siRNAs) resulting from successive DCL-catalyzed processing from the end of a double-stranded RNA substrate originating from the RDR of an AGO-catalyzed cleaved RNA at a micro RNA target site. Plant tasiRNAs have been synthesized to produce synthetic tasiRNAs (syn-tasiRNAs) targeting viral RNAs that confer viral resistance. In this study, we engineered syn-tasiRNAs to target potato virus Y (PVY) infection by replacing five native siRNAs of TAS1c with 210-bp fragments from the coat protein (CP) region of the PVY genome. The results showed that the transient expression of syn-tasiR-CPpvy2 in Nicotiana benthamiana (N. benthamiana) plants conferred antiviral resistance, supported by the absence of PVY infection symptoms and viral accumulation. This indicated that syn-tasiR-CPpvy2 successfully targeted and silenced the PVY CP gene, effectively inhibiting viral infection. syn-tasiR-CPpvy1 displayed attenuated symptoms and decreased viral accumulation in these plants However, severe symptoms of PVY infection and a similar amount of viral accumulation as the control were observed in plants expressing syn-tasiR-CPpvy3. syn-tasiR-CPpvy/pvx, which targets both PVY and potato virus X (PVX), was engineered using a single precursor. After the transient expression of syn-tasiR-CPpvy/pvx3 and syn-tasiR-CPpvy/pvx5 in N. benthamiana, the plants were resistant to both PVY and PVX. These results suggested that engineered syn-tasiRNAs could not only specifically induce antiviral resistance against one target virus but could also be designed for multi-targeted silencing of different viruses, thereby preventing complex virus infection in plants.
... accessed on 5 March 2023). The EST database was used to predict the target genes of miR-NAs by Allen et al. (2005) [32] and Schwab (2005) [33]. The prediction of miRNA target genes followed the following rules: the mismatch between miRNA and the target should not exceed four bases, the number of mismatched bases between miRNA and target gene should be less than two, there should be no adjacent mismatch between miRNA and target gene at positions 2-12, and the minimum free energy for miRNA matching with the target gene should be ≥75%. ...
... accessed on 5 March 2023). The EST database was used to predict the target genes of miR-NAs by Allen et al. (2005) [32] and Schwab (2005) [33]. The prediction of miRNA target genes followed the following rules: the mismatch between miRNA and the target should not exceed four bases, the number of mismatched bases between miRNA and target gene should be less than two, there should be no adjacent mismatch between miRNA and target gene at positions 2-12, and the minimum free energy for miRNA matching with the target gene should be ≥75%. ...
Ticks are an important type of pathogen transmission vector, and pathogens not only cause serious harm to livestock but can also infect humans. Because of the roles that ticks play in disease transmission, reducing tick pathogen infectivity has become increasingly important and requires the identification and characterization of these pathogens and their interaction mechanisms. In this study, we determined the miRNA expression profile of Hemaphysalis longicornis infected with Theileria orientalis, predicted the target genes of miRNAs involved in this infection process, and investigated the role of miRNA target recognition during host–pathogen interactions. The results showed that longipain is a target gene of miR-5309, which was differentially expressed at different developmental stages and in various tissues in the control group. However, the miR-5309 level was reduced in the infection group. Analysis of the interaction between miRNA and the target gene showed that miR-5309 negatively regulated the expression of the longipain protein during the infection of H. longicornis with T. orientalis. To verify this inference, we compared longipain with the blocking agent orientalis. In this study, the expression of longipain was upregulated by the inhibition of miR-5309 in ticks, and the ability of the antibody produced by the tick-derived protein to attenuate T. orientalis infection was verified through animal immunity and antigen–antibody binding tests. The results showed that expression of the longipain + GST fusion protein caused the cattle to produce antibodies that could be successfully captured by ticks, and cellular immunity was subsequently activated in the ticks, resulting in a subtractive effect on T. orientalis infection. This research provides ideas for the control of ticks and tickborne diseases and a research basis for studying the mechanism underlying the interaction between ticks and pathogens.
... The tasiRNAs are derived from non-protein-coding TAS transcripts that are capped as well as poly-adenylated and contain a binding site mostly for 22 nt miRNAs [156]. The miRNA cleavage products are stabilized by the Suppressor of Gene Silencing 3 (SGS3) and converted into a dsRNA form by RDR6 [157,158]. The dsRNA intermediate is then processed by DCL4 and dedicated dsRNA-Binding Protein 4 (DRB4) to phased 21 nt siRNAs in a 'head-to-tail' phased pattern. ...
... The dsRNA intermediate is then processed by DCL4 and dedicated dsRNA-Binding Protein 4 (DRB4) to phased 21 nt siRNAs in a 'head-to-tail' phased pattern. The transitive siRNAs are incorporated into AGO-RISC for targeting complementary sequences [157][158][159][160]. In Arabidopsis thaliana, eight tasiRNAproducing loci have been identified that fall into four TAS groups (TAS1-TAS4). ...
Viruses are silent enemies that intrude and take control of the plant cell's machinery for their own multiplication. Infection by viruses and the resulting damage is still a major challenge in the agriculture sector. Plants have the capability to fight back, but the ability of viruses to mutate at a fast rate helps them to evade the host's response. Therefore, classical approaches for introgressing resistance genes by breeding have obtained limited success in counteracting the virus menace. Genetic modification (GM)-based strategies have been successful in engineering artificial resistance in plants. Several different approaches based on pathogen-derived resistance, antisense constructs, hairpin RNAs, double-stranded RNA, etc., have been used to enhance plants' resistance to viruses. Recently, genome editing (GE) strategies mainly involving the CRISPR/Cas-mediated modifications are being used for virus control. In this review, we discuss the developments and advancements in GM-and GE-based methods for tackling viral infection in plants.
... There are three predominant protein families in biogenesis pathway of phasiRNAs: DICER -LIKE ( DCL ) , ARGONA UTE ( AGO ) and RNA DEPENDENT RNA polymerase ( RDR ) proteins ( 14 ,15 ) . Firstly, miRNAs with AGO proteins induce site-specific cleavages on the primary precursors of phasiRNAs ( PHAS ) (16)(17)(18) . Once triggered by miRNAs, the primary PHAS transcripts are converted into double-stranded RNAs through RDR6. ...
... Three of the identified 21 nt PHAS loci were TAS3 genes as shown in Figure 1 A and C. In addition, we found one additional TAS3 gene by aligning conserved tasiRNAs in Arabidopsis and rice to genome of tea plant (Additional file 1: Supplementary Table S8 ). TAS3a was triggered by csn-miR390b-5p at two loci (Figure 1 A, E and J) and encoded two conserved tasiARFs, named as TAS3a_D8(+) and TAS3a_D7(+) (the blue and green parts in Figure 1A, respectively), by following the nomenclature of tasiRNAs ( 16 ). Similarly, the csn-miR390b-5p was also targeting TAS3b at two complementary sites (Figure 1 B, F and J). ...
Phased secondary small interfering RNAs (phasiRNAs) in plants play important roles in regulating genome stability, plant development and stress adaption. Camellia sinensis var. assamica has immense economic, medicinal and cultural significance. Howe ver, there are still no studies of phasiRNAs and their putative functions in this valuable plant. We identified 476 and 43 PHAS loci which generated 4290 twenty one nucleotide (nt) and 264 twenty four nt phasiRNAs, respectively. Moreover, the analysis of degradome revealed more than 35000 potential targets for these phasiRNAs. We identified several conserved 21 nt phasiRNA generation pathways in tea plant, including miR390 → TAS3, miR482 / miR2118 → NB-LRR, miR393 → F-box, miR828 → MYB / TAS4, and miR7122 → PPR in this study. Furthermore, we found that some transposase and plant mobile domain genes could generate phasiRNAs. Our results show that phasiRNAs target genes in the same family in cis- or trans-manners, and different members of the same gene family may generate the same phasiRNAs. The phasiRNAs, generated by transposase and plant mobile domain genes, and their targets, suggest that phasiRNAs may be involved in the inhibition of transposable elements in tea plant. To summarize, these results provide a comprehensive view of phasiRNAs in Camellia sinensis var. assamica.
... Three miRNA families, miR160, miR167 and miR390, have been identified to confer plant growth via targeting or regulating different ARFs. In Arabidopsis, miR160 targets three ARFs genes, ARF10, ARF16 and ARF17 [43]; miR167 regulates ARF6 and ARF8 by mediating gene silencing [44]; miR390 negatively regulates the expression of ARF2, ARF3, and ARF4 by triggering TAS3-tasiRNAs [45][46][47]. Additionally, miR165/166 and miR172 were also found to be involved in other phytohormone signaling, interacting with auxin signaling. miR165/166 was proved to play critical roles in ABA homeostasis [48] and auxin signaling [49]. ...
Brace root architecture is a critical determinant of maize’s stalk anchorage and nutrition uptake, influencing root lodging resistance, stress tolerance, and plant growth. To identify the key microRNAs (miRNAs) in control of maize brace root growth, we performed small RNA sequencing using brace root samples at emergence and growth stages. We focused on the genetic modulation of brace root development in maize through manipulation of miR390 and its downstream regulated auxin response factors (ARFs). In the present study, miR167, miR166, miR172, and miR390 were identified to be involved in maize brace root growth in inbred line B73. Utilizing short tandem target mimic (STTM) technology, we further developed maize lines with reduced miR390 expression and analyzed their root architecture compared to wild-type controls. Our findings show that STTM390 maize lines exhibit enhanced brace root length and increased whorl numbers. Gene expression analyses revealed that the suppression of miR390 leads to upregulation of its downstream regulated ARF genes, specifically ZmARF11 and ZmARF26, which may significantly alter root architecture. Additionally, loss-of-function mutants for ZmARF11 and ZmARF26 were characterized to further confirm the role of these genes in brace root growth. These results demonstrate that miR390, ZmARF11, and ZmARF26 play crucial roles in regulating maize brace root growth; the involved complicated molecular mechanisms need to be further explored. This study provides a genetic basis for breeding maize varieties with improved lodging resistance and adaptability to diverse agricultural environments.
... According to a model by Harvey et al. (86), the RNA-silencing defense of the plant against an infecting virus has several levels, with AGO1 being part of a first layer and AGO2 part of a second. In uninfected plants, the expression of AGO1 is usually higher than that of AGO2 (87), and there is evidence that miR403loaded AGO1/RISC downregulate AGO2 mRNA expression post-transcriptionally (88). In virus-infected N. benthamiana, an increase in AGO2 levels can be detected (89), and one model to explain this is that viral suppressors of RNA silencing, VSRs, sequester miR403 and thus override the suppression of AGO2 expression by AGO1 (57). ...
RNA-mediated crop protection increasingly becomes a viable alternative to agrochemicals that threaten biodiversity and human health. Pathogen-derived double-stranded dsRNAs are processed into small interfering RNAs (siRNAs), which can then induce silencing of target RNAs, e.g. viral genomes. However, with currently used dsRNAs, which largely consist of undefined regions of the target RNAs, silencing is often ineffective: processing generates siRNA pools that contain only a few functionally effective siRNAs (here called esiRNAs). Using a recently developed in vitro screen that reliably identifies esiRNAs from siRNA pools, we identified esiRNAs against Cucumber Mosaic Virus (CMV), a devastating plant pathogen. Topical application of esiRNAs to plants resulted in highly effective protection against massive CMV infection. However, optimal protection was achieved with newly designed multivalent effective dsRNAs (edsRNAs), which contain the sequences of several esiRNAs and are preferentially processed into precisely these esiRNAs. The esiRNA components can attack one or more target RNAs at different sites, be active in different silencing complexes and provide cross-protection against different viral variants, important properties for combating rapidly mutating pathogens such as CMV. esiRNAs and edsRNAs have thus been established as a new class of RNA actives that significantly increase the efficacy and specificity of RNA-mediated plant protection.
... This antiviral response would occur mainly because viruses would effectively repress AGO1 (Harvey et al. 2011;Várallyay and Havelda 2013). Under normal conditions, AGO2 is in turn regulated post transcriptionally by miR403 loaded into AGO1 (Allen et al. 2005). Until recently, there has been limited information available regarding the regulation of the miR403/AGO2 system during viral infections (Xu et al. 2016;Pertermann et al. 2018;Diao et al. 2019). ...
Plant argonaute (AGO) proteins—chiefly AGO1 and 2—restrict viral infections. AGO1/2 also participate in developmental processes and are tightly regulated by microRNAs. Researchers have conducted extensive studies on the regulatory loop involving miR168/AGO1 in viral infections, though comparatively less attention has been given to the miR403/AGO2 system. Here, we simultaneously studied both regulatory systems in Arabidopsis plants infected with turnip mosaic virus (TuMV). TuMV simultaneously altered both miR168 and miR403 precursors as well as their mature forms at medium to late stages of infection. While TuMV decreased miRNA precursor molecules, it induced the overaccumulation of mature miRNA forms, without evidence of concomitant transcriptional alteration. The AGO1 protein remained at basal levels, whereas the AGO2 protein overaccumulated. The application of exogenous salicylic acid (SA) in healthy plants resulted in elevated AGO2 mRNA levels. Conversely, this hormone did not induce any significant changes in either AGO1 mRNA levels or those of miRs 168 and 403. This response is coherent with previous results, which showed enhanced levels of SA under TuMV infection and the partially differential sensitivity that AGO proteins have against this defense hormone. Our results also highlight the key role of AGO2 in leaves as an antiviral molecule and demonstrate the different responsiveness of the AGO1/miR168 and AGO2/miR403 systems regarding TuMV infection and SA response. Taken together, the results presented here are in line with previous reports studying abiotic and biotic impacts on microRNA biogenesis and AGO-dependent antiviral defense and further expand the knowledge of the miR403/AGO2 regulatory system.
... Following the method described by Fan et al. (2023), a combination of RNAhybrid (v2.1.2) + svm_light (v6.01) (Krüger and Rehmsmeier, 2006), Miranda (v3.3a) (Enright et al., 2003), and TargetScan (v7.0) (Allen et al., 2005) software was used to predict the target mRNAs of the DEmilRNAs, with default parameters set for each of the aforementioned software. Subsequently, the shared target mRNAs were considered the final targets with high confidence. ...
In the present study, small RNA (sRNA) data from Ascosphaera apis were filtered from sRNA-seq datasets from the gut tissues of A. apis-infected Apis mellifera ligustica worker larvae, which were combined with the previously gained sRNA-seq data from A. apis spores to screen differentially expressed milRNAs (DEmilRNAs), followed by trend analysis and investigation of the DEmilRNAs in relation to significant trends. Additionally, the interactions between the DEmilRNAs and their target mRNAs were verified using a dual-luciferase reporter assay. In total, 974 A. apis milRNAs were identified. The first base of these milRNAs was biased toward U. The expression of six milRNAs was confirmed by stem–loop RT-PCR, and the sequences of milR-3245-y and milR-10285-y were validated using Sanger sequencing. These miRNAs grouped into four significant trends, with the target mRNAs of DEmilRNAs involving 42 GO terms and 120 KEGG pathways, such as the fungal-type cell wall and biosynthesis of secondary metabolites. Further investigation demonstrated that 299 DEmilRNAs (novel-m0011-3p, milR-10048-y, bantam-y, etc.) potentially targeted nine genes encoding secondary metabolite-associated enzymes, while 258 (milR-25-y, milR-14-y, milR-932-x, etc.) and 419 (milR-4561-y, milR-10125-y, let-7-x, etc.) DEmilRNAs putatively targeted virulence factor-encoded genes and nine genes involved in the MAPK signaling pathway, respectively. Additionally, the interaction between ADM-B and milR-6882-x, as well as between PKIA and milR-7009-x were verified. Together, these results not only offer a basis for clarifying the mechanisms underlying DEmilRNA-regulated pathogenesis of A. apis and a novel insight into the interaction between A. apis and honey bee larvae, but also provide candidate DEmilRNA–gene axis for further investigation.
... In this study, 7556 and 5649 target genes were respectively predicted for 116 known miRNAs and 106 novel miRNAs by the online software TargetFinder (v5.8) (https://targetfinder.org/ (accessed on 13 November 2022)) ( Table S4) [66]. In order to explore the regulators involved in low-temperature resistance mediated by grafting, the target genes of cme-miR156b, cme-miR156f and chr07_30026 were identified. ...
Grafting is widely used to improve the resistance to abiotic stresses in cucurbit plants, but the effect and molecular mechanism of grafting on cold stress are still unknown in melon. In this study, phenotypic characteristics, physiological indexes, small-RNA sequencing and expression analyses were performed on grafted plants with pumpkin rootstock (PG) and self-grafted plants (SG) to explore the mechanism of changed cold tolerance by grafting in melon. Compared with SG plants, the cold tolerance was obviously enhanced, the malondialdehyde (MDA) content was significantly decreased and the activities of antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; peroxidase, POD) were significantly increased in PG plants. Depend on differentially expressed miRNA (DEM) identification and expression pattern analyses, cme-miR156b, cme-miR156f and chr07_30026 were thought to play a key role in enhancing low-temperature resistance resulting from grafting. Subsequently, 24, 37 and 17 target genes of cme-miR156b, cme-miR156f and chr07_30026 were respectively predicted, and 21 target genes were co-regulated by cme-miR156b and cme-miR156f. Among these 57 unique target genes, the putative promoter of 13 target genes contained the low-temperature responsive (LTR) cis-acting element. The results of qRT-PCR indicated that six target genes (MELO3C002370, MELO3C009217, MELO3C018972, MELO3C016713, MELO3C012858 and MELO3C000732) displayed the opposite expression pattern to their corresponding miRNAs. Furthermore, MELO3C002370, MELO3C016713 and MELO3C012858 were significantly downregulated in cold-resistant cultivars and upregulated in cold-sensitive varieties after cold stimulus, and they acted as the key negative regulators of low-temperature response in melon. This study revealed three key miRNAs and three putative target genes involved in the cold tolerance of melon and provided a molecular basis underlying how grafting improved the low-temperature resistance of melon plants.
... The PdRDR-6 protein may be required for the production of trans-acting siRNAs (tasiRNAs) because it cleavages the dsRNA. The PdDCL-4 protein may synthesize the dsRNA from the miRNA-cleavage end, resulting in 21-nucleotide siRNA duplexes formation [92][93][94]. More specifically, the identified PIWI domain contains structural similarities with ribonuclease-H enzymes, which indicates that it may be responsible for cleaving the target mRNA [95]. ...
... Among the conserved miRNA families in the Asterids, miR160 was only identified in the C. canephora subgenome whereas the miR828 family is only found in the C. eugenioides subgenome. The miR173 family, known in A. thaliana for triggering the biogenesis of both trans-acting small interfering RNAs (tasiRNAs) TAS1 and TAS2 (Allen et al., 2005), is not present in C. arabica. The families having the greatest numbers of loci were miR482, miR395, miR169, miR167, and miR171 (Table S2) whereas the most accumulated families were, in decreasing order, miR166, miR396, miR482, miR319, and miR8155 ( Figure S1). A. thaliana has no miR482/miR2118 (Zhu et al., 2013), which is the most abundant in terms of the number of loci in C. arabica. ...
Noncoding and coding RNAs are key regulators of plant growth, development, and stress responses. To investigate the types of transcripts accumulated during the vegetative to reproductive transition and floral development in the Coffea arabica L., we sequenced small RNA libraries from eight developmental stages, up to anthesis. We combined these data with messenger RNA and PARE sequencing of two important development stages that marks the transition of an apparent latent to a rapid growth stage. In addition, we took advantage of multiple in silico tools to characterize genomic loci producing small RNAs such as phasiRNAs, miRNAs, and tRFs. Our differential and co‐expression analysis showed that some types of small RNAs such as tRNAs, snoRNAs, snRNAs, and phasiRNAs preferentially accumulate in a stage‐specific manner. Members of the miR482/miR2118 superfamily and their 21‐nucleotide phasiRNAs originating from resistance genes show a robust co‐expression pattern that is maintained across all the evaluated developmental stages. Finally, the majority of miRNAs accumulate in a family stage‐specific manner, related to modulated hormonal responses and transcription factor expression.
... The PdRDR-6 protein may be required for the production of trans-acting siRNAs (tasiRNAs) because it cleavages the dsRNA. The PdDCL-4 protein may synthesize the dsRNA from the miRNA-cleavage end, resulting in 21-nucleotide siRNA duplexes formation [92][93][94]. More specifically, the identified PIWI domain contains structural similarities with ribonuclease-H enzymes, which indicates that it may be responsible for cleaving the target mRNA [95]. ...
Background
Dates contain various minerals that are essential for good health. The major RNA interference (RNAi) gene families play a vital role in plant growth and development by controlling the expression of protein-coding genes against different biotic and abiotic stresses. However, these gene families for date palm are not yet studied. Therefore, this study has explored major RNAi genes and their characteristics in date palm.
Results
We have identified 4 PdDCLs, 7 PdAGOs, and 3 PdRDRs as RNAi proteins from the date palm genome by using AtRNAi genes as query sequences in BLASTp search. Domain analysis of predicted RNAi genes has revealed the Helicase_C, Dicer_dimer, PAZ, RNase III, and Piwi domains that are associated with the gene silencing mechanisms. Most PdRNAi proteins have been found in the nucleus and cytosol associated with the gene silencing actions. The gene ontology (GO) enrichment analysis has revealed some important GO terms including RNA interference, dsRNA fragmentation, and ribonuclease_III activity that are related to the protein-coding gene silencing mechanisms. Gene regulatory network (GRN) analysis has identified PAZ and SNF2 as the transcriptional regulators of PdRNAi genes. Top-ranked 10 microRNAs including Pda-miR156b, Pda-miR396a, Pda-miR166a, Pda-miR167d, and Pda-miR529a have been identified as the key post-transcriptional regulators of PdRNAi genes that are associated with different biotic/abiotic stresses. The cis -acting regulatory element analysis of PdRNAi genes has detected some vital cis -acting elements including ABRE, MBS, MYB, MYC, Box-4, G-box, I-box, and STRE that are linked with different abiotic stresses.
Conclusion
The results of this study might be valuable resources for the improvement of different characteristics in date palm by further studies in wet-lab.
... The PdRDR-6 protein may be required for the production of trans-acting siRNAs (tasiRNAs) because it cleavages the dsRNA. The PdDCL-4 protein may synthesize the dsRNA from the miRNA-cleavage end, resulting in 21-nucleotide siRNA duplexes formation [92][93][94]. More specifically, the identified PIWI domain contains structural similarities with ribonuclease-H enzymes, which indicates that it may be responsible for cleaving the target mRNA [95]. ...
Background Dates contain various minerals that are essential for good health. The major RNA interference (RNAi) gene families play a vital role in plant growth and development by controlling the expression of protein-coding genes against different biotic and abiotic stresses. However, these gene families for date palm are not yet studied. Therefore, this study has explored major RNAi genes and their characteristics in date palm. Results We have identified 4 PdDCLs, 7 PdAGOs, and 3 PdRDRs as RNAi proteins from the date palm genome by using AtRNAi genes as query sequences in BLASTp search. Domain analysis of predicted RNAi genes has revealed the Helicase_C, Dicer_dimer, PAZ, RNase III, and Piwi domains that are associated with the gene silencing mechanisms. Most PdRNAi proteins have been found in the nucleus and cytosol associated with the gene silencing actions. The gene ontology (GO) enrichment analysis has revealed some important GO terms including RNA interference, dsRNA fragmentation, and ribonuclease_III activity that are related to the protein-coding gene silencing mechanisms. Gene regulatory network (GRN) analysis has identified PAZ and SNF2 as the transcriptional regulators of PdRNAi genes. Top-ranked 10 microRNAs including Pda-miR156b, Pda-miR396a, Pda-miR166a, Pda-miR167d, and Pda-miR529a have been identified as the key post-transcriptional regulators of PdRNAi genes that are associated with different biotic/abiotic stresses. The cis-acting regulatory element analysis of PdRNAi genes has detected some vital cis-acting elements including ABRE, MBS, MYB, MYC, Box-4, G-box, I-box, and STRE that are linked with different abiotic stresses. Conclusion The results of this study might be valuable resources for the improvement of different characteristics in date palm by further studies in wet-lab.
... By interacting with INDUCER OF CBF EXPRESSION1 (CsICE1), CsUBC24 disturbs stomate function on the anther surface, which inhibits anther dehiscence [38]. In transgenic plants, increasing miR399 transcripts reduces UBC24 transcripts [39]. Under freezing stress, overexpression of tae-miR399 ultimately decreases the expression of AtUBC24, inhibiting the degradation of ICE1 (Inducer of CBF expression 1), which increases the expression of genes involved in the C-REPEAT BINDING FACTOR (CBF) signaling pathway [40]. ...
MiR399 plays an important role in plant growth and development. The objective of the present study was to elucidate the evolutionary characteristics of the MIR399 gene family in grapevine and investigate its role in stress response. To comprehensively investigate the functions of miR399 in grapevine, nine members of the Vvi-MIR399 family were identified based on the genome, using a miRBase database search, located on four chromosomes (Chr 2, Chr 10, Chr 15, and Chr 16). The lengths of the Vvi-miR399 precursor sequences ranged from 82 to 122 nt and they formed stable stem–loop structures, indicating that they could produce microRNAs (miRNAs). Furthermore, our results suggested that the 2 to 20 nt region of miR399 mature sequences were relatively conserved among family members. Phylogenetic analysis revealed that the Vvi-MIR399 members of dicots (Arabidopsis, tomato, and sweet orange) and monocots (rice and grapevine) could be divided into three clades, and most of the Vvi-MIR399s were closely related to sweet orange in dicots. Promoter analysis of Vvi-MIR399s showed that the majority of the predicted cis-elements were related to stress response. A total of 66.7% (6/9) of the Vvi-MIR399 promoters harbored drought, GA, and SA response elements, and 44.4% (4/9) of the Vvi-MIRR399 promoters also presented elements involved in ABA and MeJA response. The expression trend of Vvi-MIR399s was consistent in different tissues, with the lowest expression level in mature and young fruits and the highest expression level in stems and young leaves. However, nine Vvi-MIR399s and four target genes showed different expression patterns when exposed to low light, high light, heat, cold, drought, and salt stress. Interestingly, a putative target of Vvi-MIR399 targeted multiple genes; for example, seven Vvi-MIR399s simultaneously targeted VIT_213s0067g03280.1. Furthermore, overexpression of Vvi_MIR399e and Vvi_MIR399f in Arabidopsis enhanced tolerance to drought compared with wild-type (WT). In contrast, the survival rate of Vvi_MIR399d-overexpressed plants were zero after drought stress. In conclusion, Vvi-MIR399e and Vvi-MIR399f, which are related to drought tolerance in grapevine, provide candidate genes for future drought resistance breeding.
... One such defense mechanism is through reprogramming of gene expression through miRNAs [17]. Plant miRNAs regulate physiological processes such as auxin signal transduction, leaf morphogenesis, flowering time, and embryo development [18,19]. A growing number of studies support the important role of miRNAs in coordinating important agronomic traits in crops. ...
Background
Alfalfa (Medicago sativa. L) is one of the best leguminous herbage in China and even in the world, with high nutritional and ecological value. However, one of the drawbacks of alfalfa is its sensitivity to dry conditions, which is a global agricultural problem. The objective of this study was to investigate the regulatory effects of endogenous nitric oxide (NO) on endogenous hormones and related miRNAs in alfalfa seedling leaves under drought stress. The effects of endogenous NO on endogenous hormones such as ABA, GA3, SA, and IAA in alfalfa leaves under drought stress were studied. In addition, high-throughput sequencing technology was used to identify drought-related miRNAs and endogenous NO-responsive miRNAs in alfalfa seedling leaves under drought stress.
Result
By measuring the contents of four endogenous hormones in alfalfa leaves, it was found that endogenous NO could regulate plant growth and stress resistance by inducing the metabolism levels of IAA, ABA, GA3, and SA in alfalfa, especially ABA and SA in alfalfa. In addition, small RNA sequencing technology and bioinformatics methods were used to analyze endogenous NO-responsive miRNAs under drought stress. It was found that most miRNAs were enriched in biological pathways and molecular functions related to hormones (ABA, ETH, and JA), phenylpropane metabolism, and plant stress tolerance.
Conclusion
In this study, the analysis of endogenous hormone signals and miRNAs in alfalfa leaves under PEG and PEG + cPTIO conditions provided an important basis for endogenous NO to improve the drought resistance of alfalfa at the physiological and molecular levels. It has important scientific value and practical significance for endogenous NO to improve plant drought resistance.
... In Arabidopsis, cleavage of transcripts by a small RNA can result in in-phase generation of 21-nt secondary siRNAs by DICER-LIKE (DCL) 4 after production of dsRNA by RNA-DEPENDENT RNA POLYMERASE (RDR) 6 (Vazquez et al. 2004; Allen et al. 2005). Such in-phase generation of 21-nt siRNAs was also detected in natural RNA silencing and co-suppression of the CHS-A gene in petunia (Kasai et al. 2013). ...
Lack of pigmentation in seed coats of soybean is caused by natural RNA silencing of the chalcone synthase (CHS) genes. This phenomenon is an evolutionary consequence of structural changes in DNA that resulted in the production of double-stranded RNAs (dsRNAs) that trigger RNA degradation. Here we determined that a mutant with pigmented seed coats derived from a cultivar that lacked the pigmentation had a deletion between DNA regions ICHS1 and a cytochrome P450 gene; the deletion included GmIRCHS, a candidate gene that triggers CHS RNA silencing via production of CHS dsRNAs. We also characterized CHS short interfering RNAs (siRNAs) produced in the wild-type seed coats that had CHS RNA silencing. Phased 21-nt CHS siRNAs were detected in all 21 phases and were widely distributed in exon 2 of CHS7, which indicates commonality in the pattern of RNA degradation in natural CHS RNA silencing between distantly related species. These results with the similarities in the rearrangements found in spontaneous mutants suggest that the structural organization that generates dsRNAs that trigger phased siRNA production is vulnerable to further structural changes, which eventually abolish the induction of RNA silencing.
... TargetFinder software was used to predict potential targets of miRNA (Allen et al. 2005). BLAST software was used to compare the predicted sequences of target genes with NR (ftp:// ftp. ...
MiRNAs play an important role in regulating plant growth and immune response. Mosaic diseases are recognized as the most important plant diseases in the world, and mosaic symptoms are recovery tissues formed by plants against virus infection. However, the mechanism of the formation of mosaic symptoms remains elusive. In this study, two typical mosaic systems consisting of Nicotiana tabacum-cucumber mosaic virus (CMV) and N. tabacum-tobacco mosaic virus (TMV) were used to investigate the relevance of miRNAs to the appearance of mosaic symptoms. The results of miRNA-seq showed that there were significant differences in miRNA abundance between dark green tissues and chlorotic tissues in mosaic leaves caused by the infection of CMV or TMV. Compared with healthy tissues, miRNA expression was significantly increased in chlorotic tissues, but slightly increased in dark green tissues. Three miRNAs, namely miR1919, miR390a, and miR6157, were identified to be strongly up-regulated in chlorotic tissues of both mosaic systems. Results of overexpressing or silencing of the three miRNAs proved that they were related to chlorophyll synthesis, auxin response, and small GTPase-mediated immunity pathway, which were corresponding to the phenotype, physiological parameters and susceptibility of the chlorotic tissues in mosaic leaves. Besides, the newly identified novel-miRNA48, novel-miRNA96 and novel-miRNA103 may also be involved in this formation of mosaic symptoms. Taken together, our results demonstrated that miR1919, miR390a and miR6157 are involved in the formation of mosaic symptoms and plant antiviral responses, providing new insight into the role of miRNAs in the formation of recovery tissue and plant immunity.
... Homologs of the majority of these genes have been found throughout angiosperms, as well as in non-seed plants (Sarojam et al., 2010). In addition to the role of these genes, posttranscriptional regulation plays an important part in the formation of leaf polarity, with important roles for miRNA165, miRNA166 and tasiR-ARF (Emery et al., 2003;Allen et al., 2005;Li et al., 2005) in the regulation of several of the genes listed above. The involvement of microRNAs in the regulation of dorsiventrality suggests that genes from the ARGONAUTE (AGO) family are relevant to this process, since they are required for the microRNA silencing of target mRNA through the formation of the RISC complex (Kidner and Martienssen, 2005;Liu et al., 2011). ...
Megaphylls, present in the majority of vascular plants, show in many plant lineages an abaxial-adaxial polarity in their dorsoventral axis. This polarity commonly translates into different tissues developing on each side of the leaf blade. This is important because it promotes better photosynthetic efficiency as related to light absorption and gas exchange. Many researchers have studied the molecular bases of the emergence of leaf abaxial-adaxial polarity, showing that it is produced by the interaction and differential expression of particular genes and other molecules. However, until now, it is still unclear if the molecular components documented thus far are sufficient to explain the emergence of leaf polarity. In this work, we integrated the available experimental data to construct a graph of the Gene Regulatory Network (GRN) involved in the formation of abaxial-adaxial polarity in the leaf primordium of Arabidopsis thaliana. This graph consisted of 21 nodes and 47 regulations. We extracted the main components of the graph to obtain a Minimum Network consisting of six genes and 22 possible regulations. Then, we used the Boolean network (BN) formalism to describe the dynamics of this Minimum Network. We identified 1905 distinct BNs that comprised the regulations of the Minimum Network and exclusively generated the two attractors representing the abaxial and adaxial cell types. This highlights the fact that most graphs, including our network, can describe experimentally observed behaviors with many BN dynamics. By performing mutant simulations and robustness analysis, we found that two of the 1905 BNs better reproduce experimentally available information. To produce the expected attractors, both BNs predict the same missing regulations, which we propose should be experimentally analyzed to confirm their existence. Interestingly, these two BNs have low robustness to perturbations compared with previously analyzed GRNs. This was an unexpected result since abaxial-adaxial polarity is a robust biological trait, which suggests more components or regulations of the network are missing.
... The candidate targets were identified in the degradome sequences for both known and novel miRNAs using two software tools: the ACGT101-DEG program (LC Sciences, Houston, TX, USA) and the CleaveLand 3.0 software package [31]. Meanwhile, the miRNAs identified were aligned to unique reads from the degradome sequence, and these alignments were scored according to established criteria for plant miRNA/target pairings [32]. Specifically, alignments with a score of no more than four, and the presence of at least one raw read at the cleavage site, were considered as candidate target transcripts. ...
Drought-induced forest death has become a global phenomenon, which is hindering the development of sustainable forestry. Polyploidy breeding has been considered as an effective method of genetic improvement for tree stress resistance. However, the response mechanisms of tetraploid poplars to drought stress are unclear. In this study, based on high-throughput sequencing of transcriptome, small RNA, and degradome for these samples, which selected three genotypes of tetraploid poplars and their counterpart diploids for drought stress and rewatering trial in the experiment, we performed multi-omics analyses to investigate the distinction in drought resistance between tetraploid and diploid. A total of 3391 differentially expressed genes (DEGs) were found from the Dro-Di vs. CK-Di, 3753 DEGs from the Re-Di vs. Dro-Di, 3857 DEGs from the Dro-Te vs. CK-Te, and 4177 DEGs from the Re-Te vs. Dro-Te. Of the above DEGs, 1646 common-DEGs were identified significantly related to drought-stress response, 2034 common-DEGs related to rewater response, 158 and 114 common-DEGs showed opposite expression patterns between diploid and tetraploid, implying that these DEGs might play important roles in response to drought stress as a result of differences in ploidy. Additionally, 586 known miRNAs and 72 novel miRNAs were identified through analysis of 18 small RNA libraries, among which eight common-miRNAs were significantly related to drought-stress response, and four were related to rewater response. The degradome sequencing analysis revealed that 154 target transcripts for 24 drought-stress-associated differentially expressed miRNAs (DEmiRs), and 90 for 12 rewatering-associated DEmiRs were identified in the tetraploid based on both degradome and TargetFinder analyses. These findings provide valuable information for further functional characterization of genes and miRNAs in response to drought stress in Populus polyploidy, and potentially contribute to drought-resistant breeding of polypoid in the future.
... siRNAs are similar to miRNAs in size and biogenesis, but they primarily silence gene expression through the RNA interference (RNAi) pathway (Dana et al., 2017;Whitehead et al., 2009). Currently, ta-siRNAs (trans-acting-small interfering RNAs) and pha-siRNAs (phased secondary small interfering RNAs) have been identified as important factors in plant development (Allen et al., 2005;Liu et al., 2020). Notably, miR390 has been found to promote leaf polarity through a ta-siRNA-dependent pathway . ...
... Mac-miR528 targets many genes encoding copper-containing proteins and polyphenol oxidase (PPO), and is downregulated in cold stress 56 . It is reported that three specialized miRNAs (miR173, miR390 and miR828) have been identified and well characterized in Arabidopsis [80][81][82] . Only miR390 is present in MaACO7 and MaACO9. ...
Ethylene is a gaseous phytohormone involved in plants’ growth and developmental processes, including seed germination, root initiation, fruit ripening, flower and leaf senescence, abscission, and stress responses. Ethylene biosynthesis (EB) gene analysis in response to nitrogen (N) and potassium (K) stress has not yet been conducted in Musa acuminata (banana) roots. The genome mining of banana (Musa acuminata L.) revealed 14 putative 1-aminocyclopropane-1-carboxylate synthase (ACS), 10 1-aminocyclopropane-1-carboxylate oxidase (ACO), and 3 Ethylene overproducer 1 (ETO1) genes. ACS, ACO, and ETO1 proteins possessed amino acid residues ranging from 422–684, 636–2670, and 893–969, respectively, with molecular weight (Mw) ranging from 4.93–7.55 kD, 10.1–8.3 kD and 10.1–10.78 kD. The number of introns present in ACS, ACO, and ETO1 gene sequences ranges from 0–14, 1–6, and 0–6, respectively. The cis-regulatory element analysis revealed the presence of light-responsive, abscisic acid, seed regulation, auxin-responsive, gibberellin element, endosperm-specific, anoxic inducibility, low-temperature responsiveness, salicylic acid responsiveness, meristem-specific and stress-responsive elements. Comprehensive phylogenetic analyses ACS, ACO, and ETO1 genes of Banana with Arabidopsis thaliana revealed several orthologs and paralogs assisting in understanding the putative functions of these genes. The expression profile of Musa acuminata genes in root under normal and low levels of nitrogen and potassium shows that MaACS14 and MaACO6 expressed highly at normal nitrogen supply. MaACS1 expression was significantly upregulated at low potassium levels, whereas, MaACO6 gene expression was significantly downregulated. The functional divergence and site-specific selective pressures on specific gene sequences of banana have been investigated. The bioinformatics-based genome-wide assessment of the family of banana attempted in the present study could be a significant step for deciphering novel ACS, ACO, and ETO1 genes based on genome-wide expression profiling.
... For target gene identification, both known miRNAs and newly predicted miRNAs were used. For target gene prediction, the miRNA sequences and the transcriptome data generated in this study were combined with TargetFinder software (Allen et al., 2005). Furthermore, differential abundance analysis between sample groups was conducted using edgeR (Robinson et al., 2010) with specific parameters of |log 2 (fold-change)| ≥ 0.58 and P-value ≤ 0.05. ...
Dendrocalamus brandisii (Munro) Kurz is a sympodial bamboo species with inimitable taste and flavorful shoots. Its rapid growth and use as high‐quality material make this bamboo species highly valued for both food processing and wood applications. However, genome information for D. brandisii is lacking, primarily due to its polyploidy and large genome size. Here, we assembled a high‐quality genome for hexaploid D. brandisii, which comprises 70 chromosomes with a total size of 2,756 Mb, using long‐read HiFi sequencing. Furthermore, we accurately separated the genome into its three constituent subgenomes. We used Oxford Nanopore Technologies long reads to construct a transcriptomic dataset covering 15 tissues for gene annotation to complement our genome assembly, revealing differential gene expression and post‐transcriptional regulation. By integrating metabolome analysis, we unveiled that well‐balanced lignin formation, as well as abundant flavonoid and fructose contents, contribute to the superior quality of D. brandisii shoots. Integrating genomic, transcriptomic, and metabolomic datasets provided a solid foundation for enhancing bamboo shoot quality and developing efficient gene‐editing techniques. This study should facilitate research on D. brandisii and enhance its use as a food source and wood material by providing crucial genomic resources.
... The miRNAs with false discovery rates (FDR) < 0.05 and |log 2 FC| > 1 were considered differentially expressed miRNAs. Based on the identified known and novel miRNAs and the tea plant genome sequence information, target gene prediction was performed using theTargetFinder software (v1.6, -c3) [31]. The functional annotation of the target genes was performed using the National Center for Biotechnology Information (NCBI) non-redundant protein (Nr) database [32], Clusters of Orthologous Groups of proteins (KOG/COG) database [33,34], the Protein family (Pfam) [35], a manually annotated and reviewed protein sequence (Swiss-Prot) database [36], the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [37] and Gene Ontology (GO) [38]. ...
Anadequate selenium (Se) intake can enhance human immunity and prevent diseases development. About one billion people in the world have varying degrees of Se deficiency in the world. Organic Se from tea infusion is the most easily absorbed and utilized Se form by the human body. Therefore the production of tea plants rich in Se is an effective way to increase Se dietary intake, but there are few studies on the involvement and functions of miRNAs in the responses of tea plants after Se treatment. MicroRNAs (miRNAs) are endogenous (non-coding) single-stranded RNAs that play crucial roles in regulating plant nutrient element acquisition and accumulation. Physiological analysis discovered that the total Se content in tea plant roots markedly increased under 0.05 mmol·L−1 selenite treatment, with no toxicity symptoms in the leaves and roots. To screen the miRNAs responsive to Se treatment in tea plants, miRNA libraries were constructed from the tea cultivar “Echa 1”. Using high-throughput sequencing, 455 known miRNAs and 203 novel miRNAs were identified in this study. In total, 13 miRNAs were selected that were differentially expressed in tea plants’ roots under 0.05 mmol·L−1 selenite treatments. Gene Ontology enrichment analysis revealed that the target genes of the differentially expressed miRNAs mainly belonged to the metabolic process, membrane, and catalytic activity ontologies. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis suggested that beta-alanine, taurine, hypotaurine, and sulfur metabolism were the most enriched pathways among the differentially expressed miRNAs, implying their involvement in Se accumulation and tolerance in tea plants. Further characterization of the data revealed that the number of novel miRNAs was comparable to that of known miRNAs, indicating that novel miRNAs significantly contributed to the regulation of Se accumulation in tea plant roots. Thisstudy lays the foundation for further research on the regulatory mechanisms underlying Se accumulation and tolerance in tea plants, providing targets to molecular breeding strategies for improving tea nutritional properties.
... Loss of miR159 leads to an increase in miR156 levels throughout shoot development, delaying plant growth, while overexpression of miR159 slightly accelerates plant growth. The primary mechanism by which miR159 inhibits miR156 is by the targeting of MYB33, a transcription factor with an R2R3 MYB domain, by miR159 [20][21][22][23]. Despite significant downregulation of miR159, loss of MYB33 still results in a premature flowering pheno-Supplementary Materials: The following supporting information can be downloaded at: https:// ...
Mulberry plants (Morus alba) have leaf shapes, ranging from unlobed to lobed, which are crucial for yield, growth, and adaptability, indicating their ability to adapt to their environment. Competing endogenous RNAs (ceRNAs) constitute a web of RNAs within the organism’s transcriptional regulatory system, including protein-coding genes (mRNAs), microRNAs (miRNAs), longnon-coding RNAs (lncRNAs), circular RNAs (circRNAs), and others. In this study, samples for ceRNA sequencing were categorized into two groups: whole leaves and lobed leaves, each group with three replicates. In addition, we isolated, cloned, and characterized the precursor miRNA (miR156x) from the leaves of M. alba. miR156x precursor had a length of 107 base pairs and a minimum folding free energy of 50.27 kcal/mol. We constructed a pCAMBIA-35S-GUS-miR156x dual overexpression vector and established a transient transformation system for mulberry. At an optimal transformation solution (OD600 = 0.7), the GUS gene showed a higher expression in the leaves of transiently transformed mulberry with miR156x overexpression, four days after transformation, while the target genes ofmiR156x had decreased expression in the same leaves. Investigations into the transgenic mulberry plants uncovered various modifications to physio-chemical parameters including POD, SOD, PRO,MDA, soluble proteins and sugars, and chlorophyl content. miRNAs in the plants were found to act as negative regulators of gene expression in response to changes in leaf shape regulation, which was confirmed in vitro using dual-luciferase reporter assays. Subsequently, we cloned Maspl3 in vitro and conducted GST-Pull down assays, obtaining multiple proteins that interacted with the Maspl3gene. This indicates that the miR156x/Maspl3/MSTRG.25812.1 regulatory module contributes to the differences in mulberry leaf shape.
... Targets prediction was performed by blast [54], the piRNA sequences were compared to the genomic sequences using blastn software (version 2.2.25), and only those that were precisely matched and complementary to each other were retained (three mismatches were allowed). Then, each sequence that could be targeted by the piRNAs was scored to predict the piRNA target loci and target mRNAs, the targets of piRNA expressed differently among different samples were analyzed statistically, and the Union set was used for enrichment analysis. ...
piRNAs are a class of small non-coding RNAs that play essential roles in modulating gene expression and abundant biological processes. To decode the piRNA-regulated larval response of western honeybees (Apis mellifera) to Ascosphaera apis infection, the expression pattern of piRNAs in Apis mellifera ligustica larval guts after A. apis inoculation was analyzed based on previously obtained high-quality small RNA-seq datasets, followed by structural characterization, target prediction, regulatory network investigation, and functional dissection. Here, 504, 657, and 587 piRNAs were respectively identified in the 4-, 5-, and 6-day-old larval guts after inoculation with A. apis, with 411 ones shared. These piRNAs shared a similar length distribution and first base bias with mammal piRNAs. Additionally, 96, 103, and 143 DEpiRNAs were detected in the 4-, 5-, and 6-day-old comparison groups. Targets of the DEpiRNAs were engaged in diverse pathways such as the phosphatidylinositol signaling system, inositol phosphate metabolism, and Wnt signaling pathway. These targets were involved in three energy metabolism-related pathways, eight development-associated signaling pathways, and seven immune-relevant pathways such as the Jak-STAT signaling pathway. The expression trends of five randomly selected DEpiRNAs were verified using a combination of RT-PCR and RT-qPCR. The effective overexpression and knockdown of piR-ame-945760 in A. apis-infected larval guts were achieved by feeding a specific mimic and inhibitor. Furthermore, piR-ame-945760 negatively regulated the expression of two target immune mRNAs, SOCS5 and ARF1, in the larval gut during the A. apis infection. These findings indicated that the overall expression level of piRNAs was increased and the expression pattern of piRNAs in larval guts was altered due to the A. apis infection, DEpiRNAs were putative regulators in the A. apis-response of A. m. ligustica worker larvae. Our data provide not only a platform for the functional investigation of piRNAs in honeybees, especially in bee larvae, but also a foundation for illuminating the piRNA-involved mechanisms underlying the host response to the A. apis infection.
... MiRNAs pair with their target genes and induce gene silencing through either transcript cleavage or translation inhibition (Achkar et al., 2016;Voinnet, 2009;Yu et al., 2017). In some cases, after the induced cleavage, miRNAs induce the generation of secondary phased siRNAs (phasiRNAs) from their target genes (Allen et al., 2005;Liu et al., 2020;Yoshikawa et al., 2005). PhasiRNAs are involved, at the post-transcriptional level similar to miRNAs, in diverse biological processes in plants (Liu et al., 2020). ...
MicroRNA482/2118 (miR482/2118) is a 22‐nt miRNA superfamily, with conserved functions in disease resistance and plant development. It usually instigates the production of phased small interfering RNAs (phasiRNAs) from its targets to expand or reinforce its silencing effect. Using a new high‐quality reference genome sequence and comprehensive small RNA profiling, we characterized a newly evolved regulatory pathway of miR482/2118 in litchi. In this pathway, miR482/2118 cleaved a novel noncoding trans ‐acting gene ( LcTASL1 ) and triggered phasiRNAs to regulate the expression of gibberellin (GA) receptor gene GIBBERELLIN INSENSITIVE DWARF1 ( GID1) in trans ; another trans ‐acting gene LcTASL2 , targeted by LcTASL1 ‐derived phasiRNAs, produced phasiRNAs as well to target LcGID1 to reinforce the silencing effect of LcTASL1 . We found this miR482/2118‐ TASL ‐ GID1 pathway was likely involved in fruit development, especially the seed development in litchi. In vivo construction of the miR482a‐ TASL ‐ GID1 pathway in Arabidopsis could lead to defects in flower and silique development, analogous to the phenotype of gid1 mutants. Finally, we found that a GA‐responsive transcription factor, LcGAMYB33, could regulate LcMIR482/2118 as a feedback mechanism of the sRNA‐silencing pathway. Our results deciphered a lineage‐specifically evolved regulatory module of miR482/2118, demonstrating the high dynamics of miR482/2118 function in plants.
... [72], RNAhybrid (v.2.1.2) + svm_light (v.6.01) [73,74], and TargetFind [75], were employed to predict the DEmiRNAs targeted by DElncRNAs as well as the DEmRNAs targeted by DEmiRNAs. Further, the DElncRNA-DEmiRNA-DEmRNA regulatory network was constructed based on the target-binding relationships and then visualized using the Cytoscape software, v.3.2.1 [76]. ...
Long non-coding RNAs (lncRNAs) are crucial modulators in a variety of biological processes, such as gene expression, development, and immune defense. However, little is known about the function of lncRNAs in the development of Asian honey bee (Apis cerana) larval guts. Here, on the basis of our previously obtained deep-sequencing data from the 4-, 5-, and 6-day-old larval guts of A. cerana workers (Ac4, Ac5, and Ac6 groups), an in-depth transcriptome-wide investigation was conducted to decipher the expression pattern, regulatory manners, and potential roles of lncRNAs during the developmental process of A. cerana worker larval guts, followed by the verification of the relative expression of differentially expressed lncRNAs (DElncRNAs) and the targeting relationships within a competing endogenous RNA (ceRNA) axis. In the Ac4 vs. Ac5 and Ac5 vs. Ac6 comparison groups, 527 and 498 DElncRNAs were identified, respectively, which is suggestive of the dynamic expression of lncRNAs during the developmental process of larval guts. A cis-acting analysis showed that 330 and 393 neighboring genes of the aforementioned DElncRNAs were, respectively involved in 29 and 32 functional terms, such as cellular processes and metabolic processes; these neighboring genes were also, respectively engaged in 246 and 246 pathways such as the Hedgehog signaling pathway and the Wnt signaling pathway. Additionally, it was found that 79 and 76 DElncRNAs as potential antisense lncRNAs may, respectively, interact with 72 and 60 sense-strand mRNAs. An investigation of competing endogenous RNA (ceRNA) networks suggested that 75 (155) DElncRNAs in the Ac4 vs. Ac5 (Ac5 vs. Ac6) comparison group could target seven (5) DEmiRNAs and further bind to 334 (248) DEmRNAs, which can be annotated to 33 (29) functional terms and 186 (210) pathways, including 12 (16) cellular- and humoral-immune pathways (lysosome pathway, necroptosis, MAPK signaling pathway, etc.) and 11 (10) development-associated signaling pathways (Wnt, Hippo, AMPK, etc.). The RT-qPCR detection of five randomly selected DElncRNAs confirmed the reliability of the used sequencing data. Moreover, the results of a dual-luciferase reporter assay were indicative of the binding relationship between MSTRG.11294.1 and miR-6001-y and between miR-6001-y and ncbi_107992440. These results demonstrate that DElncRNAs are likely to modulate the developmental process of larval guts via the regulation of the source genes’ transcription, interaction with mRNAs, and ceRNA networks. Our findings not only yield new insights into the developmental mechanism underlying A. cerana larval guts, but also provide a candidate ceRNA axis for further functional dissection.
... The molecular mechanism of RISC recognition is classified into 'one-hit pathway' and 'two-hit pathway' according to the number of RISC binding sites (Fig. 2). In one-hit pathway, the precursors are recognized by RISC composed of a 22-nt miRNA and AGO1 (43)(44)(45)(46). Analyses using TAS2 transcripts as a model showed that this binding site is subject to cleavage by RISC, but SGS3 interacts with RISC bound to TAS transcripts to hold the two fragments together after cleavage (47). ...
Many organisms produce secondary small interfering RNAs (siRNAs) that are triggered by primary small RNAs to regulate various biological processes. Plants have evolved several types of secondary siRNA biogenesis pathways that play important roles in development, stress responses and defense against viruses and transposons. The critical step of these pathways is the production of double-stranded RNAs by RNA-dependent RNA polymerases. This step is normally tightly regulated, but when its control is released, secondary siRNA production is initiated. In this article, we will review the recent advances in secondary siRNA production triggered by microRNAs encoded in the genome and siRNAs derived from invasive nucleic acids. In particular, we will focus on the factors, events, and RNA/DNA elements that promote or inhibit the early steps of secondary siRNA biogenesis.
... The Needle program in the EMBOSS package was used to extract all sequences matching the sequences of the 432 miRNAs obtained from miRNA sequencing. Arrays were scored according to plant miRNA-target interaction criteria (Allen et al. 2005). The fraction could not exceed the set threshold, and preservation of the 10th nt at the 5 end of the degradome sequence paired with miRNA was ensured. ...
The majority of woody plants are able to form ectomycorrhizal (ECM) symbioses with fungi. During symbiotic development, plants undergo a complex re-programming process involving a series of physiological and morphological changes. MicroRNAs (miRNAs) are important components of the regulatory network underlying symbiotic development. To elucidate the mechanisms of miRNAs and miRNA-mediated mRNA cleavage during symbiotic development, we conducted high-throughput sequencing of small RNAs and degradome tags from roots of Populus tomentosa inoculated with Cenococcum geophilum. This process led to the annotation of 51 differentially expressed miRNAs between non-mycorrhizal and mycorrhizal roots of P. tomentosa, including 13 novel miRNAs. Increased or decreased accumulation of several novel and conserved miRNAs in ECM roots, including miR162, miR164, miR319, miR396, miR397, miR398, novel-miR44 and novel-miR47, suggests essential roles for these miRNAs in ECM formation. The degradome analysis identified root transcripts as miRNA-mediated mRNA cleavage targets, which was confirmed using real-time quantitative PCR. Several of the identified miRNAs and corresponding targets are involved in arbuscular mycorrhizal symbioses. In summary, increased or decreased accumulation of specific miRNAs and miRNA-mediated cleavage of symbiosis-related genes indicate that miRNAs play important roles in the regulatory network underlying symbiotic development.
... All resulting reads of 'query' mRNA were reverse complemented and aligned to the miRNA identified in this study. Alignments where the degradome sequence position coincident with the tenth or eleventh nucleotide of miRNA were retained, and were scored (score ≤ 4) according to a previously described scheme developed for plant miRNA/target pairings [78]. The target was selected and categorized as 0, 1, 2, 3, or 4, as described by Sun et al. [40]. ...
Background
Neo-tetraploid rice lines exhibit high fertility and strong heterosis and harbor novel specific alleles, which are useful germplasm for polyploid rice breeding. However, the mechanism of the fertility associated with miRNAs remains unknown. In this study, a neo-tetraploid rice line, termed Huaduo21 (H21), was used. Cytological observation and RNA-sequencing were employed to identify the fertility-related miRNAs in neo-tetraploid rice.
Results
H21 showed high pollen fertility (88.08%), a lower percentage of the pollen mother cell (PMC) abnormalities, and lower abnormalities during double fertilization and embryogenesis compared with autotetraploid rice. A total of 166 non-additive miRNAs and 3108 non-additive genes were detected between H21 and its parents. GO and KEGG analysis of non-additive genes revealed significant enrichments in the DNA replication, Chromosome and associated proteins, and Replication and repair pathways. Comprehensive multi-omics analysis identified 32 pairs of miRNA/target that were associated with the fertility in H21. Of these, osa-miR408-3p and osa-miR528-5p displayed high expression patterns, targeted the phytocyanin genes, and were associated with high pollen fertility. Suppression of osa-miR528-5p in Huaduo1 resulted in a low seed set and a decrease in the number of grains. Moreover, transgenic analysis implied that osa-MIR397b-p3, osa-miR5492, and osa-MIR5495-p5 might participate in the fertility of H21.
Conclusion
Taken together, the regulation network of fertility-related miRNAs-targets pairs might contribute to the high seed setting in neo-tetraploid rice. These findings enhance our understanding of the regulatory mechanisms of pollen fertility associated with miRNAs in neo-tetraploid rice.
... Recent studies have indicated that lncRNAs are capable of absorbing target miRNAs and indirectly regulating downstream gene expression via ceRNA networks [74,75]. The potential targeting relationships between immune-defense-related DElncRNA and miRNAs, as well as miRNAs and mRNA, were predicted with RNAhybrid (V2.1.2) [76], MiRanda (V3.3a) [77], and TargetScan software (version 8.0) [78]. On the basis of the predicted targeting relationships, the DElncRNA-DEmiRNA and DELncRNA-miRNA-mRNA regulatory networks were constructed and then visualized by using Cytoscape v.3.6.1 software [79]. ...
Long non-coding RNAs (lncRNAs) play an essential part in controlling gene expression and a variety of biological processes such as immune defense and stress-response. However, whether and how lncRNAs regulate responses of Apis cerana larvae to Ascosphaera apis invasion has remained unclear until now. Here, the identification and structural analysis of lncRNAs in the guts of A. cerana worker larvae were conducted, and the expression profile of larval lncRNAs during the A. apis infection process was then analyzed, followed by an investigation of the regulatory roles of differentially expressed lncRNAs (DElncRNAs) in the host response. In total, 76 sense lncRNAs, 836 antisense lncRNAs, 184 intron lncRNAs, 362 bidirectional lncRNAs, and 2181 intron lncRNAs were discovered in the larval guts. Additionally, 30 known and 9 novel lncRNAs were potential precursors for 36 and 11 miRNAs, respectively. In the three comparison groups, 386, 351, and 272 DElncRNAs were respectively identified, indicating the change in the overall expression pattern of host lncRNAs following the A. apis invasion. Analysis of cis-acting effect showed that DElncRNAs in the 4-, 5-, and 6-day-old comparison groups putatively regulated 55, 30, and 20 up- and down-stream genes, respectively, which were involved in a series of crucial functional terms and pathways, such as MAPK signaling pathway, and cell process. Analysis showed that 31, 8, and 11 DElncRNAs as potential antisense lncRNAs may interact with 26, 8, and 9 sense-strand mRNAs. Moreover, investigation of the competing endogenous RNA (ceRNA) network indicated that 148, 283, and 257 DElncRNAs were putatively regulated. The expression of target genes by targeting corresponding DEmiRNAs included those associated with antioxidant enzymes and immune responses. These results suggested that DElncRNAs played a potential part in the larval guts responding to the A. apis infection through a cis-acting manner and ceRNA mechanisms. Our findings deepen our understanding of interactions between A. cerana larvae and A. apis and offer a basis for clarifying the DElncRNA-mediated mechanisms underlying the host response to fungal invasion.
... The epigenetic response during heat stress has been thoroughly examined by researchers. It involves the conversion of trans-acting siRNA precursor 1 (TAS1) into double-stranded RNA by RDR6 by means of a miR173 guide strand, which is further directed into a series of 21 nucleotides (trans-acting small interfering RNAs ) ta-siRNAs (Allen et al. 2005). ...
Plants have evolved to adapt and grow in hot and cold climatic conditions. Some also adapt to daily and seasonal temperature
changes. Epigenetic modifcations play an important role in regulating plant tolerance under such conditions. DNA methyla�tion and post-translational modifcations of histone proteins infuence gene expression during plant developmental stages
and under stress conditions, including cold and heat stress. While short-term modifcations are common, some modifcations
may persist and result in stress memory that can be inherited by subsequent generations. Understanding the mechanisms
of epigenomes responding to stress and the factors that trigger stress memory is crucial for developing climate-resilient
agriculture, but such an integrated view is currently limited. This review focuses on the plant epigenetic stress memory dur�ing cold and heat stress. It also discusses the potential of machine learning to modify stress memory through epigenetics to
develop climate-resilient crops.
... With the help of SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6), cleaved mRNAs form dsRNAs (Yoshikawa et al. 2005). The resulting dsRNAs are then cleaved by a DICER-LIKE 4 protein (DCL4), forming phasiRNA duplexes that are distinctive and generated in phased configuration (Allen et al. 2005;Chen et al. 2010). The loci generating phased siRNAs are called PHAS loci. ...
Phased short-interfering RNAs (phasiRNAs) fine-tune various stages of growth, development, and stress responses in plants. Potato (Solanum tuberosum) tuberization is a complex process, wherein a belowground modified stem (stolon) passes through developmental stages like swollen stolon and mini-tuber before it matures to a potato. Previously, we identified several phasiRNA-producing loci (PHAS) from stolon-to-tuber transition stages. However, whether phasiRNAs mediate tuber development remains unknown. Here, we show that a gene encoding NB-ARC DOMAIN-CONTAINING DISEASE RESISTANCE PROTEIN (StRGA4; a PHAS locus) is targeted by Stu-microRNA482c to generate phasiRNAs. Interestingly, we observed that one of the phasiRNAs, referred as short-interfering RNA D29 (-) i.e. siRD29 (-), targets the gibberellin (GA) biosynthesis gene GIBBERELLIN 3-OXIDASE 3 (StGA3ox3). Since regulation of bioactive GA levels in stolons controls tuber development, we hypothesized that a gene regulatory module, Stu-miR482c-StRGA4-siRD29(-)-StGA3ox3, could govern tuber development. Through transient expression assays and small RNA-sequencing, generation of siRD29(-) and its phase was confirmed in planta. Notably, the expression of StGA3ox3 was higher in swollen stolon compared to stolon, whereas siRD29(-) showed a negative association with StGA3ox3 expression. Antisense (AS) lines of StGA3ox3 produced more tubers compared to wild-type.. As expected, StRGA4 overexpression (OE) lines had high levels of siRD29(-) and mimicked the phenotypes of StGA3ox3-AS lines, indicating the functionality of this module in potato. In vitro tuberization assays (with or without GA inhibitor) using StGA3ox3 antisense lines and overexpression lines of StGA3ox3 or StRGA4 revealed that StGA3ox3 controls the tuber-stalk development. Taken together, our findings suggest that a phasiRNA, siRD29(-), mediates the regulation of StGA3ox3 during stolon-to-tuber transitions in potato.
... The effective concentration of the library was accurately quantified using the ABI Step One Plus Real-Time PCR system. Single-ended sequencing (SE50) was performed on the HiSeq platform to obtain 50 base pair reads of qualified fragments [18]. To assemble the transcriptome sequencing data, we utilized the Trinity software (v 2.8.5) and followed the steps outlined below: a. ...
Waterlogging stress poses a significant threat to eggplants (Solanum melongena L.), causing root oxygen deficiency and subsequent plant damage. This study aims to explore the morphological changes and chlorophyll and lignin indicators of eggplant seedlings under different time points (0, 3, 6, 12, 24, 48 h) of waterlogging stress. High-throughput sequencing was used to identify differentially expressed miRNAs and mRNAs in response to waterlogging stress in eggplants. The results showed that the content of chlorophyll a significantly decreased during the early stage of waterlogging stress, while the degradation of chlorophyll b intensified with prolonged stress, and carotenoid content remained relatively stable. Additionally, this study investigated changes in root lignin, indicating its role in enhancing cell wall stability and tolerance to cope with hypoxic stress. Using DESeq2, 246 differentially expressed miRNAs were identified, among which significant changes were observed in the miR156, miR166, miR167, and miR399 families. These miRNAs may play a crucial regulatory role in eggplant’s adaptation to the hypoxic environment after waterlogging stress. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that differentially expressed genes were mainly related to cellular physiological processes, metabolic processes, and the biosynthesis of secondary metabolites, influencing the seedlings’ stress resistance under different waterlogging conditions. Furthermore, by constructing a regulatory miRNA–target gene network that pertains to eggplant’s response to waterlogging stress, we have laid the foundation for revealing the molecular mechanisms of eggplant’s response to waterlogging stress.
Abiotic and biotic stresses globally constrain plant growth and impede the optimization of crop productivity. The phytohormone auxin is involved in nearly every aspect of plant development. Auxin acts as a chemical messenger that influences gene expression through a short nuclear pathway, mediated by a family of specific DNA-binding transcription factors known as Auxin Response Factors (ARFs). ARFs thus act as effectors of auxin response and translate chemical signals into the regulation of auxin responsive genes. Since the initial discovery of the first ARF in Arabidopsis, advancements in genetics, biochemistry, genomics, and structural biology have facilitated the development of models elucidating ARF action and their contributions to generating specific auxin responses. Yet, significant gaps persist in our understanding of ARF transcription factors despite these endeavors. Unraveling the functional roles of ARFs in regulating stress response, alongside elucidating their genetic and molecular mechanisms, is still in its nascent phase. Here, we review recent research outcomes on ARFs, detailing their involvement in regulating leaf, flower, and root organogenesis and development, as well as stress responses and their corresponding regulatory mechanisms: including gene expression patterns, functional characterization, transcriptional, post-transcriptional and post- translational regulation across diverse stress conditions. Furthermore, we delineate unresolved questions and forthcoming challenges in ARF research.
MicroRNAs are known to play a crucial role in plant development and physiology and become a target for investigating the regulatory mechanism underlying plant low phosphate tolerance. ZmmiR528 has been shown to display significantly different expression levels between wild‐type and low Pi‐tolerant maize mutants. However, its functional role in maize low Pi tolerance remains unknown. In the present study, we studied the role and underlying molecular mechanism of miR528 in maize with low Pi tolerance. Overexpression of ZmmiR528 in maize resulted in impaired root growth, reduced Pi uptake capacity and compromised resistance to Pi deficiency. By contrast, transgenic maize plants suppressing ZmmiR528 expression showed enhanced low Pi tolerance. Furthermore, ZmLac3 and ZmLac5 which encode laccase were identified and verified as targets of ZmmiR528. ZmLac3 transgenic plants were subsequently generated and were also found to play key roles in regulating maize root growth, Pi uptake and low Pi tolerance. Furthermore, auxin transport was found to be potentially involved in ZmLac3‐ mediated root growth. Moreover, we conducted genetic complementary analysis through the hybridization of ZmmiR528 and ZmLac3 transgenic plants and found a favorable combination with breeding potential, namely anti‐miR528: ZmLac3OE hybrid maize, which exhibited significantly increased low Pi tolerance and markedly alleviated yield loss caused by low Pi stress. Our study has thus identified a ZmmiR528‐ ZmLac3 module regulating auxin transport and hence root growth, thereby determining Pi uptake and ultimately low Pi tolerance, providing an effective approach for low Pi tolerance improvement through manipulating the expression of miRNA and its target in maize.
The miR390-derived TAS3 trans-acting short-interfering RNAs (tasiRNAs) module represents a conserved RNA silencing pathway in the plant kingdom; however, its characterization in the bryophyte Marchantia polymorpha is limited. This study elucidated that MpDCL4 processes MpTAS3 double-stranded RNA (dsRNA) to generate tasiRNAs, primarily from the 5ʹ- and 3ʹ-ends of dsRNA. Notably, we discovered a novel tasiRNA, tasi78A, can negatively regulate a cytochrome P450 gene, MpCYP78A101. Additionally, tasi78A was abundant in MpAGO1, and transient expression assays underscored the role of tasi78A in repressing MpCYP78A101. A microRNA, miR11700, also regulates MpCYP78A101 expression. This coordinate regulation suggests a role in modulating auxin signaling at apical notches of gemma, influencing the growth and sexual organ development of M. polymorpha and emphasizing the significance of RNA silencing in MpCYP78A101 regulation. However, phylogenetic analysis identified another paralog of the CYP78 family, Mp1g14150, which may have a redundant role with MpCYP78A101, explaining the absence of noticeable morphological changes in loss-of-function plants. Taken together, our findings provide new insights into the combined regulatory roles of miR390/MpTAS3/miR11700 in controlling MpCYP78A101 and expand our knowledge about the biogenesis and regulation of tasiRNAs in M. polymorpha.
Flowering plant genomes encode four or five DICER-LIKE (DCL) enzymes that produce small interfering RNAs (siRNAs) and microRNAs which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense and endogenous gene regulation. DCL2 acts genetically redundantly with DCL4 to confer basal antiviral defense. However, DCL2 may also counteract DCL4, since knockout of DCL4 causes growth defects that are suppressed by DCL2 inactivation. Current models maintain that RNAi via DCL2-dependent siRNAs is the biochemical basis of both effects. Here, we report that DCL2-mediated antiviral resistance and growth defects cannot be explained by the silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either with specific point mutations in DCL2 or with reduced DCL2 dosage because of heterozygosity for dcl2 knockout alleles. Intriguingly, all DCL2 functions require its catalytic activity, and the penetrance of DCL2-dependent growth phenotypes in dcl4 mutants correlates with DCL2 protein levels, but not with levels of major DCL2-dependent siRNAs. We discuss this requirement and correlation with catalytic activity, but not with resulting siRNAs, in light of other findings that reveal a DCL2 function in innate immunity activation triggered by cytoplasmic double-stranded RNA.
Plant hormones, pivotal regulators of plant growth, development, and response to environmental cues, have recently emerged as central modulators of epigenetic processes governing gene expression and phenotypic plasticity. This review addresses the complex interplay between plant hormones and epigenetic mechanisms, highlighting the diverse methodologies that have been harnessed to decipher these intricate relationships. We present a comprehensive overview to understand how phytohormones orchestrate epigenetic modifications, shaping plant adaptation and survival strategies. Conversely, we explore how epigenetic regulators ensure hormonal balance and regulate the signalling pathways of key plant hormones. Furthermore, our investigation includes a search for novel genes that are regulated by plant hormones under the control of epigenetic processes. Our review offers a contemporary overview of the epigenetic-plant hormone crosstalk, emphasizing its significance in plant growth, development, and potential agronomical applications.
Plants in the Allium genus have a significant economic impact due to their production of important food and medicinal compounds. Despite their agricultural and pharmaceutical value, breeding programs for Allium species are lagging behind those of other major crop plants. MicroRNAs (miRNAs) are a group of small non-coding RNAs that regulate post-transcriptional gene expression. In order to identify distinct miRNAs and their target proteins involved in Allicin biosynthesis pathways under heat-stress conditions, available Allium species Expressed Sequence Tags (ESTs) and miRNA databases were extensively searched and bioinformatically analyzed. Three distinct miRNA candidates targeting seven genes in garlic were identified. The real-time RT-PCR analysis results revealed that miR5021 and miR565 regulate the Alliinase and Heat shock protein 70 (HSP70) genes under cold stress (4°C), while miR5184 regulates the same genes at elevated temperatures (37 and 45°C). Based on the High-Performance Liquid Chromatography (HPLC) results, the expression of the Alliinase gene was found to be higher in both stem and leaf tissues compared to the expression of miR5021 under cold stress conditions. This indicates the potential involvement of miR5021 in the biosynthetic pathway of Allicin production. Furthermore, the results of the elevated heat-stress treatment showed that the expression of the HSP70 gene under thermal stress conditions at 37°C in both stem and leaf tissue was significantly increased compared to the expression of miR5658 and miR5184 at 45°C. However the expression of the HSP70 gene in the stem tissue was significantly decreased compared to the expression of miR5658 and miR5184 genes. This experimental study aims to identify miRNAs associated with the allicin production pathway in two tissues of garlic: leaves and cloves. Among the main findings of this research, we can mention the identification of 3 miRNAs that are likely to play a role in the allicin synthesis pathway. The results of this study suggest that environmental factors may affect the biosynthesis of allicin in plants of the Allium family.
ARGONAUTE (AGO) proteins are key components of the RNA-induced silencing complex (RISC) that mediates gene silencing in eukaryotes. Small-RNA (sRNA) cargoes are selectively loaded into different members of the AGO protein family and then target complementary sequences to in-duce transcriptional repression, mRNA cleavage, or translation inhibition. Previous reviews have mainly focused on the traditional roles of AGOs in specific biological processes or on the molecular mechanisms of sRNA sorting. In this review, we summarize the biological significance of canonical sRNA loading, including the balance among distinct sRNA pathways, cross-regulation of different RISC activities during plant development and defense, and, especially, the emerging roles of AGOs in sRNA movement. We also discuss recent advances in novel non-canonical functions of plant AGOs. Perspectives for future functional studies of this evolutionarily conserved eukaryotic protein family will facilitate a more comprehensive understanding of the multi-faceted AGO proteins.
Whereas micro (mi)RNAs are considered the clean, noble side of the small RNA world, small interfering (si)RNAs are often seen as a noisy set of molecules whose barbarian acronyms reflect a large diversity of often elusive origins. 25 years after their discovery in plants, however, new classes of siRNAs are still being identified, sometimes in discrete tissues or at particular developmental stages, making the plant siRNA world substantially more complex and subtle than originally anticipated. Focusing primarily on the model Arabidopsis, we review here the plant siRNA landscape, including transposable elements (TE)-derived siRNAs, a vast array of non-TE-derived endogenous siRNAs, as well as exogenous siRNAs produced in response to invading nucleic acids such as viruses or transgenes. We primarily emphasize the extraordinary sophistication and diversity of their biogenesis and, secondarily, the variety of their known or presumed functions, including via non-cell autonomous activities, in the sporophyte, gametophyte and shortly after fertilization.
Redclaw crayfish (Cherax quadricarinatus) is a large, tropical freshwater crustacean species with considerable potential of commercial production. In recent years, infection with DIV1 in redclaw crayfish is being reported in aquaculture industries, causing high mortality and huge economic losses. However, many characteristics of this virus, including pathogenesis, transmission mechanism, and host immunity, remain largely unknown.MicroRNAs are known to play important roles in numerous biological processes, and many microRNAs are reported to be involved in the regulation of immune responses. In this study, nine-small RNA libraries were constructed using hemocytes of redclaw crayfish to characterize the differentially expressed miRNAs (DE-miRNAs) at 24 and 48 h postinfection (hpi). A total of 14 and 22 DE-miRNAs were identified in response to DIV1 infection at 24 and 48 hpi, respectively. Further, functional annotation of the predicted host target genes using GO and KEGG pathway enrichment analyses indicated that relevant biological processes and signal pathways underwent miRNA-mediated regulation after DIV1 infection. Our results enhanced the understanding of the mechanisms of miRNA-mediated regulation of immune responses under DIV1 infection in crustaceans.
Four mutant brome mosaic virus (BMV) RNA3 transcripts, bearing single or double base changes in the 3'-CCAOH terminus of the tRNA-like structure, previously characterized as being deficient in vitro with respect to aminoacylation and replication activities, have now been assayed in vivo for their ability to replicate (in the presence of transcripts of wild-type RNA1 and -2) in barley protoplasts and plants. In tests conducted with protoplasts, irrespective of the time post-infection, all four mutants were fully viable, and the relative levels of both plus and minus strand replication for each mutant were similar to that of the wild type. Inoculation of barley plants with these mutants resulted in phenotypic symptoms and viral yields that were similar to those from wild-type infections. Analysis of each mutant progeny RNA3 indicated that the altered sequence at the 3' terminus was restored to that of wild type. These observations indicate that there is a rapid turnover and correction of the 3' termini of BMV RNAs in vivo. Such correction is commensurate with the action of tRNA nucleotidyltransferase, but it differs from recombination processes that appear to be relatively infrequent for BMV RNA3. These results support the conclusion that the 3'-CCAOH termini of viral tRNA-like structures function analogously to telomeres of chromosomal DNA.
ettin (ett) mutations have pleiotropic effects on Arabidopsis flower development, causing increases in perianth organ number, decreases in stamen number and anther formation, and apical-basal patterning defects in the gynoecium. The ETTIN gene was cloned and encodes a protein with homology to DNA binding proteins which bind to auxin response elements. ETT transcript is expressed throughout stage 1 floral meristems and subsequently resolves to a complex pattern within petal, stamen and carpel primordia. The data suggest that ETT functions to impart regional identity in floral meristems that affects perianth organ number spacing, stamen formation, and regional differentiation in stamens and the gynoecium. During stage 5, ETT expression appears in a ring at the top of the floral meristem before morphological appearance of the gynoecium, consistent with the proposal that ETT is involved in prepatterning apical and basal boundaries in the gynoecium primordium. Double mutant analyses and expression studies show that although ETT transcriptional activation occurs independently of the meristem and organ identity genes LEAFY, APETELA1, APETELA2 and AGAMOUS, the functioning of these genes is necessary for ETT activity. Double mutant analyses also demonstrate that ETT functions independently of the 'b' class genes APETELA3 and PISTILLATA. Lastly, double mutant analyses suggest that ETT control of floral organ number acts independently of CLAVATA loci and redundantly with PERIANTHIA.
lin-4 encodes a small RNA that is complementary to sequences in the 3' untranslated region (UTR) of lin-14 mRNA and that acts to developmentally repress the accumulation of LIN-14 protein. This repression is essential for the proper timing of numerous events of Caenorhabditis elegans larval development. We have investigated the mechanism of lin-4 RNA action by examining the fate of lin-14 mRNA in vivo during the time that lin-4 RNA is expressed. Our results indicate that the rate of synthesis of lin-14 mRNA, its state of polyadenylation, its abundance in the cytoplasmic fraction, and its polysomal sedimentation profile do not change in response to the accumulation of lin-4 RNA. Our results indicate that association of lin-4 RNA with the 3' UTR of lin-14 mRNA permits normal biogenesis of lin-14 mRNA, and normal translational initiation, but inhibits step(s) thereafter, such as translational elongation and/or the release of stable LIN-14 protein.
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Introduction of transgene DNA may lead to specific degradation of RNAs that are homologous to the transgene transcribed sequence through phenomena named post-transcriptional gene silencing (PTGS) in plants, quelling in fungi, and RNA interference (RNAi) in animals. It was shown previously that PTGS, quelling, and RNAi require a set of related proteins (SGS2, QDE-1, and EGO-1, respectively). Here we report the isolation of Arabidopsis mutants impaired in PTGS which are affected at the Argonaute1 (AGO1) locus. AGO1 is similar to QDE-2 required for quelling and RDE-1 required for RNAi. Sequencing of ago1 mutants revealed one amino acid essential for PTGS that is also present in QDE-2 and RDE-1 in a highly conserved motif. Taken together, these results confirm the hypothesis that these processes derive from a common ancestral mechanism that controls expression of invading nucleic acid molecules at the post-transcriptional level. As opposed to rde-1 and qde-2 mutants, which are viable, ago1 mutants display several developmental abnormalities, including sterility. These results raise the possibility that PTGS, or at least some of its elements, could participate in the regulation of gene expression during development in plants.
The heterochronic gene lin-28 of the nematode Caenorhabditis elegans controls the relative timing of diverse developmental events during the animal's larval stages. lin-28 is stage-specifically regulated by two genetic circuits: negatively by the 22-nt RNA lin-4 and positively by the heterochronic gene lin-14. Here, we show that lin-28 is repressed during normal development by a mechanism that acts on its mRNA after translation initiation. We provide evidence that lin-14 inhibits a negative regulation that is independent of the lin-4 RNA and involves the gene daf-12, which encodes a nuclear hormone receptor. The lin-4-independent repression does not affect the initiation of translation on the lin-28 mRNA, and like the lin-4-mediated repression, acts through the gene's 3'-untranslated region. In addition, we find that lin-4 is not sufficient to cause repression of lin-28 if the lin-4-independent circuit is inhibited. Therefore, the lin-4-independent circuit likely contributes substantially to the down-regulation of lin-28 that occurs during normal development. The role of lin-4 may be to initiate or potentiate the lin-4-independent circuit. We speculate that a parallel lin-4-independent regulatory mechanism regulates the expression of lin-14.
In metazoans, microRNAs, or miRNAs, constitute a growing family of small regulatory RNAs that are usually 19-25 nucleotides in length. They are processed from longer precursor RNAs that fold into stem-loop structures by the ribonuclease Dicer and are thought to regulate gene expression by base pairing with RNAs of protein-coding genes. In Arabidopsis thaliana, mutations in CARPEL FACTORY (CAF), a Dicer homolog, and those in a novel gene, HEN1, result in similar, multifaceted developmental defects, suggesting a similar function of the two genes, possibly in miRNA metabolism.
To investigate the potential functions of CAF and HEN1 in miRNA metabolism, we aimed to isolate miRNAs from Arabidopsis and examine their accumulation during plant development in wild-type plants and in hen1-1 and caf-1 mutant plants. We have isolated 11 miRNAs, some of which have potential homologs in tobacco, rice, and maize. The putative precursors of these miRNAs have the capacity to form stable stem-loop structures. The accumulation of these miRNAs appears to be spatially or temporally controlled in plant development, and their abundance is greatly reduced in caf-1 and hen1-1 mutants. HEN1 homologs are found in bacterial, fungal, and metazoan genomes.
miRNAs are present in both plant and animal kingdoms. An evolutionarily conserved mechanism involving a protein, known as Dicer in animals and CAF in Arabidopsis, operates in miRNA metabolism. HEN1 is a new player in miRNA accumulation in Arabidopsis, and HEN1 homologs in metazoans may have a similar function. The developmental defects associated with caf-1 and hen1-1 mutations and the patterns of miRNA accumulation suggest that miRNAs play fundamental roles in plant development.
Micro-RNAs (miRNAs) are regulatory molecules that mediate effects by interacting with messenger RNA (mRNA) targets. Here we show that Arabidopsis thaliana miRNA 39 (also known as miR171), a 21-ribonucleotide species that accumulates predominantly in inflorescence tissues, is produced from an intergenic region in chromosome III and functionally interacts with mRNA targets encoding several members of the Scarecrow-like (SCL) family of putative transcription factors. miRNA 39 is complementary to an internal region of three SCL mRNAs. The interaction results in specific cleavage of target mRNA within the region of complementarity, indicating that this class of miRNA functions like small interfering RNA associated with RNA silencing to guide sequence-specific cleavage in a developmentally controlled manner.
Proteins of the ARGONAUTE family are important in diverse posttranscriptional RNA-mediated gene-silencing systems as well
as in transcriptional gene silencing in Drosophila and fission yeast and in programmed DNA elimination in Tetrahymena. We cloned ARGONAUTE4 (AGO4) from a screen for mutants that suppress silencing of the Arabidopsis SUPERMAN(SUP) gene. The ago4-1 mutant reactivated silentSUP alleles and decreased CpNpG and asymmetric DNA methylation as well as histone H3 lysine-9 methylation. In addition,ago4-1 blocked histone and DNA methylation and the accumulation of 25-nucleotide small interfering RNAs (siRNAs) that correspond
to the retroelement AtSN1. These results suggest that AGO4 and long siRNAs direct chromatin modifications, including histone methylation and non-CpG
DNA methylation.
Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000
T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the ∼29,454 predicted Arabidopsis genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site
bias at both the chromosome and gene levels. Insertion mutations were identified in genes that are regulated in response to
the plant hormone ethylene.
Plant microRNAs (miRNAs) show a high degree of sequence complementarity to, and are believed to guide the cleavage of, their
target messenger RNAs. Here, Ishow that miRNA172, which can base-pair with the messenger RNA of a floral homeotic gene, APETALA2, regulates APETALA2 expression primarily through translational inhibition. Elevated miRNA172 accumulation results in floral organ identity defects
similar to those in loss-of-function apetala2 mutants. Elevated levels of mutant APETALA2 RNA with disrupted miRNA172 base pairing, but not wild-type APETALA2 RNA, result in elevated levels of APETALA2 protein and severe floral patterning defects. Therefore, miRNA172 likely acts
in cell-fate specification as a translational repressor of APETALA2 in Arabidopsis flower development.
Plants with altered microRNA metabolism have pleiotropic developmental defects, but direct evidence for microRNAs regulating specific aspects of plant morphogenesis has been lacking. In a genetic screen, we identified the JAW locus, which produces a microRNA that can guide messenger RNA cleavage of several TCP genes controlling leaf development. MicroRNA-guided cleavage of TCP4 mRNA is necessary to prevent aberrant activity of the TCP4 gene expressed from its native promoter. In addition, overexpression of wild-type and microRNA-resistant TCP variants demonstrates that mRNA cleavage is largely sufficient to restrict TCP function to its normal domain of activity. TCP genes with microRNA target sequences are found in a wide range of species, indicating that microRNA-mediated control of leaf morphogenesis is conserved in plants with very different leaf forms.
The RNA interference (RNAi) pathway is initiated by processing long double-stranded RNA into small interfering RNA (siRNA).
The siRNA-generating enzyme was purified from Drosophila S2cells and consists of two stoichiometric subunits: Dicer-2(DCR-2) and a previously unknown protein that we named R2D2.
R2D2 is homologous to the Caenorhabditis elegans RNAi protein RDE-4. Association with R2D2 does not affect the enzymatic activity of DCR-2. Rather, the DCR-2/R2D2 complex,
but not DCR-2 alone, binds to siRNA and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated
silencing complex (RISC). These results indicate that R2D2 bridges the initiation and effector steps of the Drosophila RNAi pathway by facilitating siRNA passage from Dicer to RISC.
MicroRNAs (miRNAs) are approximately 21-nucleotide noncoding RNAs that have been identified in both animals and plants. Although in animals there is direct evidence implicating particular miRNAs in the control of developmental timing, to date it is not known whether plant miRNAs also play a role in regulating temporal transitions. Through an activation-tagging approach, we demonstrate that miRNA 172 (miR172) causes early flowering and disrupts the specification of floral organ identity when overexpressed in Arabidopsis. miR172 normally is expressed in a temporal manner, consistent with its proposed role in flowering time control. The regulatory target of miR172 is a subfamily of APETALA2 (AP2) transcription factor genes. We present evidence that miR172 downregulates these target genes by a translational mechanism rather than by RNA cleavage. Gain-of-function and loss-of-function analyses indicate that two of the AP2-like target genes normally act as floral repressors, supporting the notion that miR172 regulates flowering time by downregulating AP2-like target genes.
MicroRNAs (miRNAs), which function as regulators of gene expression in eukaryotes, are processed from larger transcripts by
sequential action of nuclear and cytoplasmic ribonuclease III–like endonucleases. We show that Exportin-5 (Exp5) mediates
efficient nuclear export of short miRNA precursors (pre-miRNAs) and that its depletion by RNA interference results in reduced
miRNA levels. Exp5 binds correctly processed pre-miRNAs directly and specifically, in a Ran guanosine triphosphate–dependent
manner, but interacts only weakly with extended pre-miRNAs that yield incorrect miRNAs when processed by Dicer in vitro. Thus,
Exp5 is key to miRNA biogenesis and may help coordinate nuclear and cytoplasmic processing steps.
MicroRNAs (miRNAs) are initially expressed as long transcripts that are processed in the nucleus to yield approximately 65-nucleotide (nt) RNA hairpin intermediates, termed pre-miRNAs, that are exported to the cytoplasm for additional processing to yield mature, approximately 22-nt miRNAs. Here, we demonstrate that human pre-miRNA nuclear export, and miRNA function, are dependent on Exportin-5. Exportin-5 can bind pre-miRNAs specifically in vitro, but only in the presence of the Ran-GTP cofactor. Short hairpin RNAs, artificial pre-miRNA analogs used to express small interfering RNAs, also depend on Exportin-5 for nuclear export. Together, these findings define an additional cellular cofactor required for miRNA biogenesis and function.
The Arabidopsis HYL1 gene encodes a nuclear double-stranded RNA-binding protein. A knockout mutation of the hyl1 gene is recessive and pleiotropic, causing developmental abnormalities, increasing sensitivity to abscisic acid, and reducing sensitivity to auxin and cytokinin. We report that levels of several microRNAs (miRNAs; miR159, -167, and -171) are reduced in homozygous mutant plants, and levels of two of three tested target mRNAs are elevated. Conversely, the miRNA levels are elevated in plants expressing a HYL1 cDNA from a strong promoter, and the corresponding target RNAs are reduced. These changes result from alterations in the stability of the target RNAs. However, double-stranded RNA-induced posttranscriptional gene silencing is unaffected by the hyl1 mutation. One-third to one-half of the cellular HYL1 protein is in a macromolecular complex, and a GFP-HYL1 fusion protein is found predominantly in the nucleus, although it is observed in both nucleus and cytoplasm in some cells. Within nuclei, HYL1 is associated with subnuclear bodies and ring-like structures. These observations provide evidence that the HYL1 protein is part of a nuclear macromolecular complex that is involved in miRNA-mediated gene regulation. Because hyl1 mutants show marked abnormalities in hormone responses, these results further suggest that miRNA-mediated changes in mRNA stability play a vital role in plant hormone signaling.
In both animals and plants, many developmentally important regulatory genes have complementary microRNAs (miRNAs), which suggests that these miRNAs constitute a class of developmental signalling molecules. Leaves of higher plants exhibit a varying degree of asymmetry along the adaxial/abaxial (upper/lower) axis. This asymmetry is specified through the polarized expression of class III homeodomain/leucine zipper (HD-ZIPIII) genes. In Arabidopsis, three such genes, PHABULOSA (PHB), PHAVOLUTA (PHV) and REVOLUTA (REV), are expressed throughout the incipient leaf, but become adaxially localized after primordium emergence. Downregulation of the HD-ZIPIII genes allows expression of the KANADI and YABBY genes, which specify abaxial fate. PHB, PHV and REV transcripts contain a complementary site for miRNA165 and miRNA166, which can direct their cleavage in vitro. Here we show that miRNA166 constitutes a highly conserved polarizing signal whose expression pattern spatially defines the expression domain of the maize hd-zipIII family member rolled leaf1 (rld1). Moreover, the progressively expanding expression pattern of miRNA166 during leaf development and its accumulation in phloem suggests that miRNA166 may form a movable signal that emanates from a signalling centre below the incipient leaf.
Multicellular eukaryotes produce small RNA molecules (approximately 21-24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense.
MicroRNAs (miRNAs) are endogenous 21-24-nt RNAs that can down-regulate gene expression by pairing to the messages of protein-coding genes to specify mRNA cleavage or repression of productive translation. They act within the RNA-induced silencing complex (RISC), which in animals contains a member of the Argonaute family of proteins. In the present study, we show that Arabidopsis ago1 mutants have increased accumulation of mRNAs known to be targeted for cleavage by miRNAs. In hypomorphic ago1 alleles, this compromised miRNA function occurs without a substantial change in miRNA accumulation, whereas in null alleles it is accompanied by a drop in some of the miRNAs. Therefore, AGO1 acts within the Arabidopsis miRNA pathway, probably within the miRNA-programmed RISC, such that the absence of AGO1 destabilizes some of the miRNAs. We also show that targeting of AGO1 mRNA by miR168 is needed for proper plant development, illustrating the importance of feedback control by this miRNA. Transgenic plants expressing a mutant AGO1 mRNA with decreased complementarity to miR168 overaccumulate AGO1 mRNA and exhibit developmental defects partially overlapping with those of dcl1, hen1, and hyl1 mutants showing a decrease in miRNA accumulation. miRNA targets overaccumulate in miR168-resistant plants, suggesting that a large excess of AGO1 protein interferes with the function of RISC or sequesters miRNAs or other RISC components. Developmental defects induced by a miR168-resistant AGO1 mRNA can be rescued by a compensatory miRNA that is complementary to the mutant AGO1 mRNA, proving the regulatory relationship between miR168 and its target and opening the way for engineering artificial miRNAs in plants.
Both microRNAs (miRNA) and small interfering RNAs (siRNA) share a common set of cellular proteins (Dicer and the RNA-induced silencing complex [RISC]) to elicit RNA interference. In the following work, a statistical analysis of the internal stability of published miRNA sequences in the context of miRNA precursor hairpins revealed enhanced flexibility of miRNA precursors, especially at the 5'-anti-sense (AS) terminal base pair. The same trend was observed in siRNA, with functional duplexes displaying a lower internal stability (Delta0.5 kcal/mol) at the 5'-AS end than nonfunctional duplexes. Average internal stability of siRNA molecules retrieved from plant cells after introduction of long RNA sequences also shows this characteristic thermodynamic signature. Together, these results suggest that the thermodynamic properties of siRNA play a critical role in determining the molecule's function and longevity, possibly biasing the steps involved in duplex unwinding and strand retention by RISC.
A key step in RNA interference (RNAi) is assembly of the RISC, the protein-siRNA complex that mediates target RNA cleavage. Here, we show that the two strands of an siRNA duplex are not equally eligible for assembly into RISC. Rather, both the absolute and relative stabilities of the base pairs at the 5' ends of the two siRNA strands determine the degree to which each strand participates in the RNAi pathway. siRNA duplexes can be functionally asymmetric, with only one of the two strands able to trigger RNAi. Asymmetry is the hallmark of a related class of small, single-stranded, noncoding RNAs, microRNAs (miRNAs). We suggest that single-stranded miRNAs are initially generated as siRNA-like duplexes whose structures predestine one strand to enter the RISC and the other strand to be destroyed. Thus, the common step of RISC assembly is an unexpected source of asymmetry for both siRNA function and miRNA biogenesis.
Motivation:
When running experiments that involve multiple high density oligonucleotide arrays, it is important to remove sources of variation between arrays of non-biological origin. Normalization is a process for reducing this variation. It is common to see non-linear relations between arrays and the standard normalization provided by Affymetrix does not perform well in these situations.
Results:
We present three methods of performing normalization at the probe intensity level. These methods are called complete data methods because they make use of data from all arrays in an experiment to form the normalizing relation. These algorithms are compared to two methods that make use of a baseline array: a one number scaling based algorithm and a method that uses a non-linear normalizing relation by comparing the variability and bias of an expression measure. Two publicly available datasets are used to carry out the comparisons. The simplest and quickest complete data method is found to perform favorably.
Availability:
Software implementing all three of the complete data normalization methods is available as part of the R package Affy, which is a part of the Bioconductor project http://www.bioconductor.org.
Supplementary information:
Additional figures may be found at http://www.stat.berkeley.edu/~bolstad/normalize/index.html
Sulfate uptake and ATP sulfurylase activity in the roots of Arabidopsis thaliana and Brassica napus were enhanced by S deprivation and reduced following resupply of SO42–. Similar responses occurred in split-root experiments where only a portion of the root system was S-deprived, suggesting that the regulation involves inter-organ signaling. Phloem-translocated glutathione (GSH) was identified as the likely transducing molecule responsible for regulating SO42– uptake rate and ATP sulfurylase activity in roots. The regulatory role of GSH was confirmed by the finding that ATP sulfurylase activity was inhibited by supplying Cys except in the presence of buthionine sulfoximine, an inhibitor of GSH synthesis. In direct and remote (split-root) exposures, levels of protein detected by antibodies against the Arabidopsis APS3 ATP sulfurylase increased in the roots of A. thaliana and B. napus during S starvation, decreased after SO42– restoration, and declined after feeding GSH. RNA blot analysis revealed that the transcript level of APS1, which codes for ATP sulfurylase, was reduced by direct and remote GSH treatments. The abundance of AST68 (a gene encoding an SO42– transporter) was similarly affected by altered sulfur status. This report presents the first evidence for the regulation of root genes involved in nutrient acquisition and assimilation by a signal that is translocated from shoot to root.
In RNA interference (RNAi), double-stranded RNA (dsRNA) triggers degradation of homologous messenger RNA. In many organisms, RNA-dependent RNA polymerase (RdRp) is required to initiate or amplify RNAi, but the substrate for dsRNA synthesis in vivo is not known. Here, we show that RdRp-dependent transgene silencing in Arabidopsis was caused by mutation of XRN4, which is a ribonuclease (RNase) implicated in mRNA turnover by means of decapping and 5'-3' exonucteotysis. When both XRN4 and the RdRp were mutated, the plants accumulated decapped transgene mRNA. We propose that mRNAs lacking a cap structure become exposed to RdRp to initiate or maintain RNAi.
We describe a new method (T-Coffee) for multiple sequence alignment that provides a dramatic improvement in accuracy with a modest sacrifice in speed as compared to the most commonly used alternatives. The method is broadly based on the popular progressive approach to multiple alignment but avoids the most serious pitfalls caused by the greedy nature of this algorithm. With T-Coffee we pre-process a data set of all pair-wise alignments between the sequences. This provides us with a library of alignment information that can be used to guide the progressive alignment. Intermediate alignments are then based not only on the sequences to be aligned next but also on how all of the sequences align with each other. This alignment information can be derived from heterogeneous sources such as a mixture of alignment programs and/or structure superposition. Here, we illustrate the power of the approach by using a combination of local and global pair-wise alignments to generate the library. The resulting alignments are significantly more reliable, as determined by comparison with a set of 141 test cases, than any of the popular alternatives that we tried. The improvement, especially clear with the more difficult test cases, is always visible, regardless of the phylogenetic spread of the sequences in the tests.
Sulfate uptake and ATP sulfurylase activity in the roots of Arabidopsis thaliana and Brassica napus were enhanced by S deprivation and reduced following resupply of SO4(2-). Similar responses occurred in split-root experiments where only a portion of the root system was S-deprived, suggesting that the regulation involves inter-organ signaling. Phloem-translocated glutathione (GSH) was identified as the likely transducing molecule responsible for regulating SO4(2-) uptake rate and ATP sulfurylase activity in roots. The regulatory role of GSH was confirmed by the finding that ATP sulfurylase activity was inhibited by supplying Cys except in the presence of buthionine sulfoximine, an inhibitor of GSH synthesis. In direct and remote (split-root) exposures, levels of protein detected by antibodies against the Arabidopsis APS3 ATP sulfurylase increased in the roots of A. thaliana and B. napus during S starvation, decreased after SO4(2-) restoration, and declined after feeding GSH. RNA blot analysis revealed that the transcript level of APS1, which codes for ATP sulfurylase, was reduced by direct and remote GSH treatments. The abundance of AST68 (a gene encoding an SO4(2-) transporter) was similarly affected by altered sulfur status. This report presents the first evidence for the regulation of root genes involved in nutrient acquisition and assimilation by a signal that is translocated from shoot to root.
Posttranscriptional gene silencing is a defense mechanism in plants that is similar to quelling in fungi and RNA interference in animals. Here, we describe four genetic loci that are required for posttranscriptional gene silencing in Arabidopsis. One of these, SDE1, is a plant homolog of QDE-1 in Neurospora crassa that encodes an RNA-dependent RNA polymerase. The sde1 mutation was specific for posttranscriptional gene silencing induced by transgenes rather than by viruses. We propose that the role of SDE1 is to synthesize a double-stranded RNA initiator of posttranscriptional gene silencing. According to this idea, when a virus induces posttranscriptional gene silencing, the virus-encoded RNA polymerase would produce the double-stranded RNA and SDE1 would be redundant.
Posttranscriptional gene silencing (PTGS) in plants resuits from the degradation of mRNAs and shows phenomenological similarities with quelling in fungi and RNAi in animals. Here, we report the isolation of sgs2 and sgs3 Arabidopsis mutants impaired in PTGS. We establish a mechanistic link between PTGS, quelling, and RNAi since the Arabidopsis SGS2 protein is similar to an RNA-dependent RNA polymerase like N. crassa QDE-1, controlling quelling, and C. elegans EGO-1, controlling RNAi. In contrast, SGS3 shows no significant similarity with any known or putative protein, thus defining a specific step of PTGS in plants. Both sgs2 and sgs3 mutants show enhanced susceptibility to virus, definitively proving that PTGS is an antiviral defense mechanism that can also target transgene RNA for degradation.
The phytohormone auxin has wide-ranging effects on growth and development. Genetic and physiological approaches implicate auxin flux in determination of floral organ number and patterning. This study uses a novel technique of transiently applying a polar auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA), to developing Arabidopsis flowers to further characterize the role of auxin in organogenesis. NPA has marked effects on floral organ number as well as on regional specification in wild-type gynoecia, as defined by morphological and histological landmarks for regional boundaries, as well as tissue-specific reporter lines. NPA's effects on gynoecium patterning mimic the phenotype of mutations in ETTIN, a member of the auxin response factor family of transcription factors. In addition, application of different concentrations of NPA reveal an increased sensitivity of weak ettin alleles to disruptions in polar auxin transport. In contrast, the defects found in spatula gynoecia are partially rescued by treatment with NPA. A model is proposed suggesting an apical-basal gradient of auxin during gynoecium development. This model provides a mechanism linking ETTIN's putative transcriptional regulation of auxin-responsive genes to the establishment or elaboration of tissue patterning during gynoecial development.
The 3' ends of RNAs associated with turnip crinkle virus (TCV), including subviral satellite (sat)C, terminate with the motif CCUGCCC-3'. Transcripts of satC with a deletion of the motif are repaired to wild type (wt) in vivo by RNA-dependent RNA polymerase (RdRp)-mediated extension of abortively synthesized oligoribonucleotide primers complementary to the 3' end of the TCV genomic RNA. Repair of shorter deletions, however, are repaired by other mechanisms. SatC transcripts with the 3' terminal CCC replaced by eight nonviral bases were repaired in plants by homologous recombination between the similar 3' ends of satC and TCV. Transcripts with deletions of four or five 3' terminal bases, in the presence or absence of nonviral bases, generated progeny with a mixture of wt and non-wt 3' ends in vivo. In vitro, RdRp-containing extracts were able to polymerize nucleotides in a template-independent fashion before using these primers to initiate transcription at or near the 3' end of truncated satC templates. The nontemplate additions at the 5' ends of the nascent complementary strands were not random, with a preference for consecutive identical nucleotides. The RdRp was also able to initiate transcription opposite cytidylate, uridylate, guanylate, and possibly adenylate residues without exhibiting an obvious preference, flexibility previously unreported for viral RdRp. The unexpected existence of three different repair mechanisms for TCV suggests that 3' end reconstruction is critical to virus survival.
RNA viruses use several initiation strategies to ensure that their RNAs are synthesized in appropriate amounts, have correct termini, and can be translated efficiently. Many viruses with genomes of single-stranded positive-, negative-, and double-stranded RNA initiate RNA synthesis by a de novo (primer-independent) mechanism. This review summarizes biochemical features and variations of de novo initiation in viral RNA replication.
In organisms as diverse as nematodes, trypanosomes, plants, and fungi, double-stranded RNA triggers the destruction of homologous mRNAs, a phenomenon known as RNA interference. RNA interference begins with the transformation of the double-stranded RNA into small RNAs that then guide a protein nuclease to destroy their mRNA targets.
MicroRNAs (miRNAs) are an extensive class of ~22-nucleotide noncoding RNAs thought to regulate gene expression in metazoans. We find that miRNAs are also present in plants, indicating that this class of noncoding RNA arose early in eukaryotic evolution. In this paper 16 Arabidopsis miRNAs are described, many of which have differential expression patterns in development. Eight are absolutely conserved in the rice genome. The plant miRNA loci potentially encode stem-loop precursors similar to those processed by Dicer (a ribonuclease III) in animals. Mutation of an Arabidopsis Dicer homolog, CARPEL FACTORY, prevents the accumulation of miRNAs, showing that similar mechanisms direct miRNA processing in plants and animals. The previously described roles of CARPEL FACTORY in the development of Arabidopsis embryos, leaves, and floral meristems suggest that the miRNAs could play regulatory roles in the development of plants as well as animals.
We predict regulatory targets for 14 Arabidopsis microRNAs (miRNAs) by identifying mRNAs with near complementarity. Complementary sites within predicted targets are conserved in rice. Of the 49 predicted targets, 34 are members of transcription factor gene families involved in developmental patterning or cell differentiation. The near-perfect complementarity between plant miRNAs and their targets suggests that many plant miRNAs act similarly to small interfering RNAs and direct mRNA cleavage. The targeting of developmental transcription factors suggests that many plant miRNAs function during cellular differentiation to clear key regulatory transcripts from daughter cell lineages.
Genetic studies of embryo, ovule and flower development in Arabidopsis thaliana have led to the independent isolation of different mutant alleles of a single gene (SIN1/SUS1/CAF, now renamed DCL1) that encodes a complex RNA-processing enzyme. DCL1 shows similarity to the Dicer group of genes, which are required for RNA silencing in Drosophila and Caenorhabditis. These recent findings identify a novel but conserved mechanism of post-transcriptional gene regulation that is important for development in eukaryotes.
RNA silencing phenomena were first discovered in plants, yet only the RNA interference pathway in animals has been subject to biochemical analysis. Here, we extend biochemical analysis to plant RNA silencing. We find that standard wheat germ extract contains Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs, as well as an RNA-dependent RNA polymerase activity that can convert exogenous single-stranded RNA into dsRNA. In this plant embryo extract, an endogenous microRNA (miRNA) that lacks perfect complementarity to its RNA targets nonetheless acts as a small interfering RNA. The miRNA guides an endonuclease to cleave efficiently wild-type Arabidopsis PHAVOLUTA mRNA, but not a dominant mutant previously shown to perturb leaf development. This finding supports the view that plant miRNAs direct RNAi and that miRNA-specified mRNA destruction is important for proper plant development. Thus, endonuclease complexes guided by small RNAs are a common feature of RNA silencing in both animals and plants.
When running experiments that involve multiple high density oligonucleotide arrays, it is important to remove sources of variation between arrays of non-biological origin. Normalization is a process for reducing this variation. It is common to see non-linear relations between arrays and the standard normalization provided by Affymetrix does not perform well in these situations.
We present three methods of performing normalization at the probe intensity level. These methods are called complete data methods because they make use of data from all arrays in an experiment to form the normalizing relation. These algorithms are compared to two methods that make use of a baseline array: a one number scaling based algorithm and a method that uses a non-linear normalizing relation by comparing the variability and bias of an expression measure. Two publicly available datasets are used to carry out the comparisons. The simplest and quickest complete data method is found to perform favorably.
Software implementing all three of the complete data normalization methods is available as part of the R package Affy, which is a part of the Bioconductor project http://www.bioconductor.org.
Additional figures may be found at http://www.stat.berkeley.edu/~bolstad/normalize/index.html
The molecular basis for virus-induced disease in plants has been a long-standing mystery. Infection of Arabidopsis by Turnip mosaic virus (TuMV) induces a number of developmental defects in vegetative and reproductive organs. We found that these defects, many of which resemble those in miRNA-deficient dicer-like1 (dcl1) mutants, were due to the TuMV-encoded RNA-silencing suppressor, P1/HC-Pro. Suppression of RNA silencing is a counterdefensive mechanism that enables systemic infection by TuMV. The suppressor interfered with the activity of miR171 (also known as miRNA39), which directs cleavage of several mRNAs coding for Scarecrow-like transcription factors, by inhibiting miR171-guided nucleolytic function. Out of ten other mRNAs that were validated as miRNA-guided cleavage targets, eight accumulated to elevated levels in the presence of P1/HC-Pro. The basis for TuMV- and other virus-induced disease in plants may be explained, at least partly, by interference with miRNA-controlled developmental pathways that share components with the antiviral RNA-silencing pathway.
Loss-of-function mutations of HASTY (HST) affect many different processes in Arabidopsis development. In addition to reducing the size of both roots and lateral organs of the shoot, hst mutations affect the size of the shoot apical meristem, accelerate vegetative phase change, delay floral induction under short days, adaxialize leaves and carpels, disrupt the phyllotaxis of the inflorescence, and reduce fertility. Double mutant analysis suggests that HST acts in parallel to SQUINT in the regulation of phase change and in parallel to KANADI in the regulation of leaf polarity. Positional cloning demonstrated that HST is the Arabidopsis ortholog of the importin beta-like nucleocytoplasmic transport receptors exportin 5 in mammals and MSN5 in yeast. Consistent with a potential role in nucleocytoplasmic transport, we found that HST interacts with RAN1 in a yeast two-hybrid assay and that a HST-GUS fusion protein is located at the periphery of the nucleus. HST is one of at least 17 members of the importin-beta family in Arabidopsis and is the first member of this family shown to have an essential function in plants. The hst loss-of-function phenotype suggests that this protein regulates the nucleocytoplasmic transport of molecules involved in several different morphogenetic pathways, as well as molecules generally required for root and shoot growth.
RNA-dependent RNA polymerases (RdRPs) have been implicated in posttranscriptional gene silencing (PTGS) and antiviral defense. An Arabidopsis RdRP (SDE1/SGS2) has been previously shown to be required for transgene-induced PTGS but has no general role in antiviral defense. On the other hand, we have recently shown that transgenic tobacco deficient in an inducible RdRP (NtRdRP1) activity became more susceptible to both Tobacco mosaic virus and Potato virus X. Thus, different RdRPs may have distinct roles in closely related PTGS and antiviral defense. In the present study, we analyzed roles of a newly identified Arabidopsis RdRP gene (AtRdRP1) in plant antiviral defense. AtRdRP1 encodes an RdRP closely related structurally to NtRdRP1 and is also induced by salicylic acid treatment and virus infection. A T-DNA insertion mutant for AtRdRP1 has been isolated and analyzed for possible alterations in response to viral infection. When infected by a tobamovirus and a tobravirus, the knockout mutant accumulated higher and more persistent levels of viral RNAs in both the lower, inoculated and in upper, systemically infected leaves than did wild-type plants. These results suggest that the inducible AtRdRP1 is the Arabidopsis ortholog of NtRdRP1 and plays a role in antiviral defense. Examination of short viral RNAs and silencing studies using a viral vector harboring an endogenous plant gene suggest that, while not required for virus-induced PTGS, AtRdRP1 can apparently promote turnover of viral RNAs in infected plants.
Formation of microRNA (miRNA) requires an RNaseIII domain-containing protein, termed DICER-1 in animals and DICER-LIKE1 (DCL1) in plants, to catalyze processing of an RNA precursor with a fold-back structure. Loss-of-function dcl1 mutants of Arabidopsis have low levels of miRNA and exhibit a range of developmental phenotypes in vegetative, reproductive, and embryonic tissues. In this paper, we show that DCL1 mRNA occurs in multiple forms, including truncated molecules that result from aberrant pre-mRNA processing. Both full-length and truncated forms accumulated to relatively low levels in plants containing a functional DCL1 gene. However, in dcl1 mutant plants, dcl1 RNA forms accumulated to levels several-fold higher than those in DCL1 plants. Elevated levels of DCL1 RNAs were also detected in miRNA-defective hen1 mutant plants and in plants expressing a virus-encoded suppressor of RNA silencing (P1/HC-Pro), which inhibits miRNA-guided degradation of target mRNAs. A miRNA (miR162) target sequence was predicted near the middle of DCL1 mRNA, and a DCL1-derived RNA with the properties of a miR162-guided cleavage product was identified and mapped. These results indicate that DCL1 mRNA is subject to negative feedback regulation through the activity of a miRNA.
Drosophila geneticists have uncovered roles for microRNAs in the coordination of cell proliferation and cell death during development, and in stress resistance and fat metabolism. In C. elegans, a homolog of the well-known fly developmental regulator hunchback acts downstream of the microRNAs lin-4 and let-7 in a pathway controlling developmental timing.
Shoots of all land plants have a radial pattern that can be considered to have an adaxial (central)-abaxial (peripheral) polarity. In Arabidopsis, gain-of-function alleles of PHAVOLUTA and PHABULOSA, members of the class III HD-ZIP gene family, result in adaxialization of lateral organs. Conversely, loss-of-function alleles of the KANADI genes cause an adaxialization of lateral organs. Thus, the class III HD-ZIP and KANADI genes comprise a genetic system that patterns abaxial-adaxial polarity in lateral organs produced from the apical meristem.
We show that gain-of-function alleles of REVOLUTA, another member of the class III HD-ZIP gene family, are characterized by adaxialized lateral organs and alterations in the radial patterning of vascular bundles in the stem. The gain-of-function phenotype can be obtained by changing only the REVOLUTA mRNA sequence and without changing the protein sequence; this finding indicates that this phenotype is likely mediated through an interference with microRNA binding. Loss of KANADI activity results in similar alterations in vascular patterning as compared to REVOLUTA gain-of-function alleles. Simultaneous loss-of-function of PHABULOSA, PHAVOLUTA, and REVOLUTA abaxializes cotyledons, abolishes the formation of the primary apical meristem, and in severe cases, eliminates bilateral symmetry; these phenotypes implicate these three genes in radial patterning of both embryonic and postembryonic growth.
Based on complementary vascular and leaf phenotypes of class III HD-ZIP and KANADI mutants, we propose that a common genetic program dependent upon miRNAs governs adaxial-abaxial patterning of leaves and radial patterning of stems in the angiosperm shoot. This finding implies that a common patterning mechanism is shared between apical and vascular meristems.
microRNAs (miRNAs) are widespread among eukaryotes, and studies in several systems have revealed that miRNAs can regulate expression of specific genes. Primary miRNA transcripts are initially processed to approximately 70-nucleotide (nt) stem-loop structures (pre-miRNAs), exported to the cytoplasm, further processed to yield approximately 22-nt dsRNAs, and finally incorporated into ribonucleoprotein particles, which are thought to be the active species. Here we study nuclear export of pre-miRNAs and show that the process is saturable and thus carrier-mediated. Export is sensitive to depletion of nuclear RanGTP and, according to this criterion, mediated by a RanGTP-dependent exportin. An unbiased affinity chromatography approach with immobilized pre-miRNAs identified exportin 5 as the pre-miRNA-specific export carrier. We have cloned exportin 5 from Xenopus and demonstrate that antibodies raised against the Xenopus receptor specifically block pre-miRNA export from nuclei of Xenopus oocytes. We further show that exportin 5 interacts with double-stranded RNA in a sequence-independent manner.
MicroRNAs (miRNAs) are endogenous approximately 22 nt RNAs that can play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.
Gene regulation by RNA interference requires the functions of the PAZ domain protein Argonaute. In plants, mutations in ARGONAUTE1 (AGO1) are associated with distinctive developmental defects that suggest a role for microRNA (miRNA) in organ polarity. Potential targets of miRNA regulation are the homeodomain/leucine zipper genes PHABULOSA (PHB) and PHAVOLUTA (PHV). These genes are expressed in a polar fashion in leaf primordia and are required for adaxial cell fate. Here we show that a 21-nucleotide miRNA that directs cleavage of PHB/PHV messenger RNA accumulates first in the embryonic meristem, and then in the abaxial domain of the developing leaf. miRNA distribution is disrupted by mutations in AGO1, indicating that AGO1 affects the regulation of miRNA. In addition, interactions between homeodomain/leucine zipper genes and an allelic series of ago1 indicate that miRNA acts as a signal to specify leaf polarity.
We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. Our results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 "holo-RISC" complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi.