Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation.

Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was initially reported for viral RNA, but later found also on eukaryotic transcripts, resulting in proteome diversification and protein-level modulation. Here, we report that AGO1x, an evolutionarily conserved translational readthrough isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double-stranded RNAs (dsRNAs) and consequent induction of interferon responses and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x exhibits nuclear localization in the vicinity of nucleoli. We identify AGO1x interaction with the polyribonucleotide nucleotidyltransferase 1 (PNPT1) and show that the depletion of this protein further augments dsRNA accumulation. Our study thus uncovers a novel function of an Argonaute protein in buffering the endogenous dsRNA-induced interferon responses, different than the canonical function of AGO proteins in the miRNA effector pathway. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study thus suggests a new direction for limiting tumor growth.

Antagonistic cotranscriptional regulation through ARGONAUTE1 and the THO/TREX complex orchestrates FLC transcriptional output.

Quantitative transcriptional control is essential for physiological and developmental processes in many organisms. Transcriptional output is influenced by cotranscriptional processes interconnected to chromatin regulation, but how the functions of different cotranscriptional regulators are integrated is poorly understood. The Arabidopsis floral repressor locus FLOWERING LOCUS C (FLC) is cotranscriptionally repressed by alternative processing of the antisense transcript COOLAIR. Proximal 3'-end processing of COOLAIR resolves a cotranscriptionally formed R-loop, and this process physically links to a histone-modifying complex FLD/SDG26/LD. This induces a chromatin environment locally that determines low transcription initiation and a slow elongation rate to both sense and antisense strands. Here, we show that ARGONAUTE1 (AGO1) genetically functions in this cotranscriptional repression mechanism. AGO1 associates with COOLAIR and influences COOLAIR splicing dynamics to promote proximal COOLAIR, R-loop resolution, and chromatin silencing. Proteomic analyses revealed physical associations between AGO1, subunits of RNA Polymerase II (Pol II), the splicing-related proteins-the spliceosome NineTeen Complex (NTC) and related proteins (NTR)-and the THO/TREX complex. We connect these activities by demonstrating that the THO/TREX complex activates FLC expression acting antagonistically to AGO1 in COOLAIR processing. Together these data reveal that antagonistic cotranscriptional regulation through AGO1 or THO/TREX influences COOLAIR processing to deliver a local chromatin environment that determines FLC transcriptional output. The involvement of these conserved cotranscriptional regulators suggests similar mechanisms may underpin quantitative transcriptional regulation generally.

GmDFB1, an ARM-repeat superfamily protein, regulates floral organ identity through repressing siRNA- and miRNA-mediated gene silencing in soybean.

The development of flowers in soybean (Glycine max) is essential for determining the yield potential of the plant. Gene silencing pathways are involved in modulating flower development, but their full elucidation is still incomplete. Here, we conducted a forward genetic screen and identified an abnormal flower mutant, deformed floral bud1-1 (Gmdfb1-1), in soybean. We mapped and identified the causal gene, which encodes a member of the armadillo (ARM)-repeat superfamily. Using small RNA sequencing (sRNA-seq), we found an abnormal accumulation of small interfering RNAs (siRNAs) and microRNA (miRNAs) in the Gmdfb1 mutants. We further demonstrated that GmDFB1 interacts with the RNA exosome cofactor SUPER KILLER7 (GmSKI7). Additionally, GmDFB1 interacts with the PIWI domain of ARGONAUTE 1 (GmAGO1) to inhibit the cleavage efficiency on the target genes of sRNAs. The enhanced gene silencing mediated by siRNA and miRNA in the Gmdfb1 mutants leads to the downregulation of their target genes associated with flower development. This study revealed the crucial role of GmDFB1 in regulating floral organ identity in soybean probably by participating in two distinct gene silencing pathways.

LncRNA PVT1 promotes the progression of ovarian cancer by activating TGF-ß pathway via miR-148a-3p/AGO1 axis.

Ovarian cancer is a lethal gynaecologic malignancy with poor diagnosis and prognosis. The long non-coding RNA plasmacytoma variant translocation1 (PVT1) and argonaute 1 (AGO1) are associated with carcinogenesis and chemoresistance; however, the relationship between PVT1 and AGO1 and the downstream mechanisms in ovarian cancer remains poorly known. PVT1 and AGO1 expression was assessed through RT-qPCR and Western blotting in both human tissues and cell lines. The viability and proliferation of ovarian cancer cells were determined by CCK-8 assay and TUNEL assay in vitro and immunohistochemistry in vivo. Cell invasion and migration were investigated through transwell and wound-healing assays. The roles and mechanisms of AGO1 on cell functions were further probed via gain- and loss-of-function analysis. We reveal that PVT1 expression was significantly increased in ovarian cancer tissues which is associated with advanced FIGO stage, lymph-node metastasis, poor survival rate, and high expression of AGO1. PVT1 or AGO1 knockdown significantly reduced the cell viability and increased the cell apoptosis and inhibited ovarian tumour growth and proliferation. Furthermore, we discovered that PVT1 up-regulated the expression of AGO1 and thus regulated the transforming growth factor-ß (TGF-ß) pathway to promote ovarian cancer progression through sponging miR-148a-3p. Additionally, the activation of ERK1/2, smad2 and smad4 is observed to be related to the PVT1/miR-148a-3p/AGO1/TGF-ß pathway-induced cascades. Taken together, the present study reveals that PVT1/miR-148a/AGO1 axis plays an important role in the progression of ovarian cancer and emphasize the potential as a target of value for ovarian cancer therapy.

Mechanisms ensuring robust repression of the Drosophila female germline stem cell maintenance factor Nanos via posttranscriptional regulation.

During oogenesis in the Drosophila ovary, numerous translational regulators promote the self-renewal or differentiation of stem cells. An intriguing question is how these regulators combine to execute translational regulation. Here, we study mechanisms for the posttranscriptional regulation of nos, a critical stem cell self-renewal factor in the Drosophila ovary; specifically, regulators that promote differentiation of the stem cell daughter. Previous studies showed that Bam, Bgcn, Mei-P26, and Sxl form a complex and repress nos expression through the nos 3'UTR. To further elucidate mechanistic processes of Nos translational regulation, we reconstituted nos repression in cultured Drosophila cells. We identify Ago1 and Brat as new members, and show that Ago1 acts through miRNA binding sites in the proximal region of the nos 3'UTR, whereas Sxl acts via an Sxl binding sequence in the distal region. Combining these findings with published reports, we propose that additional factors Bam, Bgcn, Mei-P26, and Brat are recruited to nos mRNAs through interaction with Ago1 and Sxl. These findings elucidate mechanisms of nos regulation by diverse translational repressors.

miR168 targets Argonaute1A mediated miRNAs regulation pathways in response to potassium deficiency stress in tomato.

BACKGROUND: Potassium (K+) is an essential ion for most plants, as it is involved in the regulation of growth and development. K+ homeostasis in plant cells has evolved to facilitate plant adaptation to K+-deficiency stress. Argonaute1 (AGO1) is regulated by miR168 to modulate the small RNA regulatory pathway by RNA silencing complex (RISC) in tomatoes. However, the role of miR168-mediated regulation of AGO1 in the context of K+ deficiency stress in tomatoes has not been elucidated yet. RESULTS: SlmiR168 and its target gene SlAGO1A were differentially expressed among low-K+-tolerant JZ34 and low-K+-sensitive JZ18 tomato plants. Transgenic tomato plants constitutively expressing pri-SlmiR168a showed stronger root system growth, better leaves development, and higher K+ contents in roots under K+-deficiency stress than those of the transgenic tomato lines expressing rSlAGO1A (SlmiR168-resistant) and the wild type (WT). Deep sequencing analysis showed that 62 known microRNAs (miRNAs) were up-regulated in 35S:rSlAGO1 compared with WT tomatoes. The same miRNAs were down-regulated in 35S:SlmiR168a compared with WT plants. The integrated analysis found 12 miRNA/mRNA pairs from the 62 miRNAs, including the root growth and cytokinin (CTK)/abscisic acid (ABA) pathways. CONCLUSIONS: The regulation mediated by SlmiR168 of SlAGO1A contributes to the plant development under low-K+ stress. Moreover, this regulation mechanism may influence downstream miRNA pathways in response to low-K+ stress through the CTK/ABA and root growth modulation pathways.

Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti.

The mosquito Aedes aegypti is a major vector of numerous viral diseases, because it requires a blood meal to facilitate egg development. The fat body, a counterpart of mammalian liver and adipose tissues, is the metabolic center, playing a key role in reproduction. Therefore, understanding of regulatory networks controlling its functions is critical, and the role of microRNAs (miRNAs) in the process is largely unknown. We aimed to explore miRNA expression and potential targets in the female fat body of Ae. aegypti, as well as their changes postblood meal (PBM). Small RNA library analysis revealed five unique miRNA patterns sequentially expressed at five sampled time points, likely responding to, and affecting, waves of upstream hormonal signals and gene expression in the same period. To link miRNA identities with downstream targets, transcriptome-wide mRNA 3' UTR interaction sites were experimentally determined at 72 h posteclosion and 24 h PBM through Argonaute 1 cross-linking and immunoprecipitation followed by high-throughput sequencing. Several target sites were validated by means of in vitro luciferase assays with wild-type and mutated 3' UTRs for six miRNA families. With established transgenic lines, consistent results were observed with spatiotemporal knockdown of miR-8 and luciferase assays. We further investigated miRNAs potentially regulating various physiological processes based on Clusters of Orthologous Groups functional categories. Hence, the present work comprehensively elucidated miRNA expression and target dynamics in the female mosquito fat body, providing a solid foundation for future functional studies of miRNA regulation during the gonadotrophic cycle.

Malus hupehensis miR168 Targets to ARGONAUTE1 and Contributes to the Resistance against Botryosphaeria dothidea Infection by Altering Defense Responses.

MicroRNA (miRNA)-mediated post-transcriptional regulation plays a fundamental role in various plant physiological processes, including responses to pathogens. MicroRNA168 has been implicated as an essential factor of miRNA pathways by targeting ARGONAUTE1 (AGO1), the core component of the RNA-induced silencing complex (RISC). A fluctuation in AGO1 expression influences various plant-pathogen interactions, and the homeostasis of AGO1 and miR168 accumulation is maintained by a complicated feedback regulatory loop. In this study, the connection between miR168 and the resistance of Malus hupehensis to Botryosphaeria dothidea is revealed. The induction of both the mature miR168 and its precursor in plants subjected to B. dothidea infection indicate the transcriptional activation of MIR168a. MIR168a promoter analysis demonstrates that the promoter can be activated by B. dothidea and salicylic acid (SA). However, the direct target of miR168, M. hupehensis ARGONAUTE1 (MhAGO1), is shown to be induced under the infection. Expression and transcription activity analysis demonstrate the transcriptional activation and the post-transcriptional suppression of MhAGO1 in response to B. dothidea infection. By inhibiting reactive oxygen species (ROS) production and enhancing SA-mediated defense responses, miR168a delays the symptom development of leaves inoculated with B. dothidea and impedes the pathogen growth, while MhAGO1 is found to have the opposite effects. Collectively, these findings suggest that the expression of miR168 and MhAGO1 in M. hupehensis in response to B. dothidea infection is regulated by a complicated mechanism. Targeting to MhAGO1, a negative regulator, miR168 plays a positive role in the resistance by alterations in diverse defense responses.

The 'how' and 'where' of plant microRNAs.

Contents 1002 I. 1002 II. 1007 III. 1010 IV. 1013 1013 References 1013 SUMMARY: MicroRNAs (miRNAs) are small non-coding RNAs, of typically 20-24 nt, that regulate gene expression post-transcriptionally through sequence complementarity. Since the identification of the first miRNA, lin-4, in the nematode Caenorhabditis elegans in 1993, thousands of miRNAs have been discovered in animals and plants, and their regulatory roles in numerous biological processes have been uncovered. In plants, research efforts have established the major molecular framework of miRNA biogenesis and modes of action, and are beginning to elucidate the mechanisms of miRNA degradation. Studies have implicated restricted and surprising subcellular locations in which miRNA biogenesis or activity takes place. In this article, we summarize the current knowledge on how plant miRNAs are made and degraded, and how they repress target gene expression. We discuss not only the players involved in these processes, but also the subcellular sites in which these processes are known or implicated to take place. We hope to raise awareness that the cell biology of miRNAs holds the key to a full understanding of these enigmatic molecules.

The legume miR1514a modulates a NAC transcription factor transcript to trigger phasiRNA formation in response to drought.

Recent studies have identified microRNAs as post-transcriptional regulators involved in stress responses. miR1514a is a legume microRNA that is induced in response to drought stress in Phaseolus vulgaris (common bean) and shows differential accumulation levels in roots during water deficit in two cultivars with different drought tolerance phenotypes. A recent degradome analysis revealed that miR1514a targets the transcripts of two NAC transcription factors (TFs), Phvul.010g121000 and Phvul.010g120700. Furthermore, expression studies and small RNA-seq data indicate that only Phvul.010g120700 generates phasiRNAs, which also accumulate under water deficit conditions. To confirm these results, we over-expressed miR1514a in transgenic hairy roots, and observed a reduced accumulation of Phvul.010g120700 and an increase in NAC-derived phasiRNAs; inhibition of miR1514a activity resulted in the opposite effect. Moreover, we determined that a NAC-derived phasiRNA associates with ARGONAUTE 1 (AGO1), suggesting that it is functional. In addition, a transcriptome analysis of transgenic hairy roots with reduced miR1514a levels revealed several differentially expressed transcripts, mainly involved in metabolism and stress responses, suggesting they are regulated by the NAC TF and/or by phasiRNAs. This work therefore demonstrates the participation of miR1514 in the regulation of a NAC transcription factor transcript through phasiRNA production during the plant response to water deficit.

A reversed framework for the identification of microRNA-target pairs in plants.

Most plant microRNAs (miRNAs) perform their repressive regulation through target cleavages. The resulting slicing sites on the target transcripts could be mapped by sequencing of the 3'-cleavage remnants, called degradome sequencing. The high sequence complementarity between miRNAs and their targets has greatly facilitated the development of the target prediction tools for plant miRNAs. The prediction results were then subjected to degradome sequencing data-based validation, through which numerous miRNA-target interactions have been extracted. However, some drawbacks are unavoidable when using this forward approach. Essentially, a known list of plant miRNAs should be obtained in advance of target prediction and validation. This becomes an obstacle to discover novel miRNAs and their targets. Here, after reviewing the current available algorithms for reverse identification of miRNA-target pairs in plants, a case study was performed by using a newly established framework with adjustable parameters. In this workflow, integration of degradome and ARGONAUTE 1-enriched small RNA sequencing data was recommended to do a relatively comprehensive and reliable search. Besides, several computational algorithms such as BLAST, target plots and RNA secondary structure prediction were used. The results demonstrated the prevalent utility of the reversed approach for uncovering miRNA-target interactions in plants.

DDX6 post-transcriptionally down-regulates miR-143/145 expression through host gene NCR143/145 in cancer cells.

In various human malignancies, widespread dysregulation of microRNA (miRNA) expression is reported to occur and affects various cell growth programs. Recent studies suggest that the expression levels of miRNAs that act as tumor suppressors are frequently reduced in cancers because of chromosome deletions, epigenetical changes, aberrant transcription, and disturbances in miRNA processing. MiR-143 and -145 are well-recognized miRNAs that are highly expressed in several tissues, but down-regulated in most types of cancers. However, the mechanism of this down-regulation has not been investigated in detail. Here, we show that DEAD-box RNA helicase 6, DDX6 (p54/RCK), post-transcriptionally down-regulated miR-143/145 expression by prompting the degradation of its host gene product, NCR143/145 RNA. In human gastric cancer cell line MKN45, DDX6 protein was abundantly expressed and accumulated in processing bodies (P-bodies). DDX6 preferentially increased the instability of non-coding RNA, NCR143/145, which encompasses the miR-143/145 cluster, and down-regulated the expression of mature miR-143/145. In human monocytic cell line THP-1, lipopolysaccharide treatment promoted the assembly of P-bodies and down-regulated the expression of NCR143/145 and its miR-143/145 rapidly. In these cells, cycloheximide treatment led to a loss of P-bodies and to an increase in NCR143/145 RNA stability, thus resulting in up-regulation of miR-143/145 expression. These data demonstrate that DDX6 contributed to the control of NCR143/145 RNA stability in P-bodies and post-transcriptionally regulated miR-143/145 expression in cancer cells.

miR168 influences phase transition, leaf epinasty, and fruit development via SlAGO1s in tomato.

In Arabidopsis thaliana, Argonaute1 (AGO1) interacts with miR168 to modulate the small RNA regulatory pathway. However, the underlying mechanism of regulation and relationship between AGO1 and miR168 is poorly understood in the cash crop Solanum lycopersicum (tomato). We previously found that SlAGO1A and SlAGO1B were cleaved by miR168 in tomato. In this study, we show that SlAGO1A and SlAGO1B accumulate in miR168-sponge transgenic plants, and that expression of miR168-resistant SlAGO1A (4m-SlAGO1A) and SlAGO1B (4m-SlAGO1B) in tomato results in a series of defects affecting growth rate, floral timing, leaves, and fruit. Accumulation of miR156 was found when 4m-SlAGO1A was at an early developmental stage compared to the wild type and original SlAGO1A transgenic plants, and miR172 was highly expressed in adult 4m-SlAGO1A compared to the controls. In addition, the expression of multiple small RNAs was altered in 4m-SlAGO1A. Taken together, our data provide novel insights into the interaction between SlAGO1s and miR168 in determining growth rate, phase change, leaf epinasty, fruit initiation and expansion, and other developmental processes in tomato.

AGO1 controls arabidopsis inflorescence architecture possibly by regulating TFL1 expression.

BACKGROUND AND AIMS: The TERMINAL FLOWER 1 (TFL1) gene is pivotal in the control of inflorescence architecture in arabidopsis. Thus, tfl1 mutants flower early and have a very short inflorescence phase, while TFL1-overexpressing plants have extended vegetative and inflorescence phases, producing many coflorescences. TFL1 is expressed in the shoot meristems, never in the flowers. In the inflorescence apex, TFL1 keeps the floral genes LEAFY (LFY) and APETALA1 (AP1) restricted to the flower, while LFY and AP1 restrict TFL1 to the inflorescence meristem. In spite of the central role of TFL1 in inflorescence architecture, regulation of its expression is poorly understood. This study aims to expand the understanding of inflorescence development by identifying and studying novel TFL1 regulators. METHODS: Mutagenesis of an Arabidopsis thaliana line carrying a TFL1::GUS (ß-glucuronidase) reporter construct was used to isolate a mutant with altered TFL1 expression. The mutated gene was identified by positional cloning. Expression of TFL1 and TFL1::GUS was analysed by real-time PCR and histochemical GUS detection. Double-mutant analysis was used to assess the contribution of TFL1 to the inflorescence mutant phenotype. KEY RESULTS: A mutant with both an increased number of coflorescences and high and ectopic TFL1 expression was isolated. Cloning of the mutated gene showed that both phenotypes were caused by a mutation in the ARGONAUTE1 (AGO1) gene, which encodes a key component of the RNA silencing machinery. Analysis of another ago1 allele indicated that the proliferation of coflorescences and ectopic TFL1 expression phenotypes are not allele specific. The increased number of coflorescences is suppressed in ago1 tfl1 double mutants. CONCLUSIONS: The results identify AGO1 as a repressor of TFL1 expression. Moreover, they reveal a novel role for AGO1 in inflorescence development, controlling the production of coflorescences. AGO1 seems to play this role through regulating TFL1 expression.

A new loss-of-function allele 28y reveals a role of ARGONAUTE1 in limiting asymmetric division of stomatal lineage ground cell.

In Arabidopsis thaliana L., stomata are produced through a series of divisions including asymmetric and symmetric divisions. Asymmetric entry division of meristemoid mother cell produces two daughter cells, the smaller meristemoid and the larger sister cell, a stomatal lineage ground cell (SLGC). Stomatal lineage ground cells can differentiate into epidermal pavement cells but have the potential to divide asymmetrically, spacing divisions, to create satellite meristemoids. Peptide ligands and TOO MANY MOUTHS (TMM) and ERECTA family receptors regulate the initiation of stomatal lineages, activity, and orientation of spacing divisions. Here, we reported that a natural mutant 28y displayed an increased stomatal density and index. Using map-based cloning, we identified mutation in ARGONAUTE1 (AGO1) as the cause of 28y phenotypes. Time-lapse tracing of stomatal lineage cells reveals that stomatal overproduction in 28y is caused by the excessive asymmetric spacing division of SLGCs. Further genetic results demonstrated that AGO1 acts downstream of TMM and negatively regulates the SPCH transcripts, but in a brassinosteroid-independent manner. Upregulation of AGAMOUS-LIKE16 (AGL16) in 28y mutants suggests that AGO1 is required to restrict AGL16-mediated stomatal spacing divisions, an miRNA pathway in addition to ligand-receptor signaling modules.

NtAGO1 positively regulates the generation and viral resistance of dark green islands in Nicotiana tabacum.

Dark green islands (DGIs) are the outcome of post-transcriptional gene silencing (PTGS) in antiviral immunity, but their characteristics related to PTGS remain largely unknown. In this study, the cucumber mosaic virus (CMV) was inoculated on Nicotiana tabacum plants to explore the PTGS features of DGIs. Our results showed that higher expressions of PTGS-associated genes, especially NtAGO1, present in DGIs. To investigate the role of NtAGO1 in the generation and the antiviral effect of DGIs, NtAGO1 was then over-expressed or knocked out in N. tabacum plants through agrobacterium-mediated genetic transformation. The results showed that more DGIs with larger areas appeared on NtAGO1 over-expressed plants, accompanied by less virus accumulation, less reactive oxygen species production, and seldom membrane damage, whereas fewer DGIs appeared on NtAGO1 knockout plants with more damage on infected plants. In addition, the NtAGO1-participated antiviral process could promote the transduction of the salicylic acid-mediated defense pathway. Taken together, our results indicate that DGIs are maintained by a stronger PTGS mechanism, and NtAGO1 positively regulates the generation and viral resistance of DGIs in N. tabacum.

Extensive Analysis of miRNA Trimming and Tailing Indicates that AGO1 Has a Complex Role in miRNA Turnover.

MicroRNAs are small regulatory RNAs involved in several processes in plants ranging from development and stress responses to defense against pathogens. In order to accomplish their molecular functions, miRNAs are methylated and loaded into one ARGONAUTE (AGO) protein, commonly known as AGO1, to stabilize and protect the molecule and to assemble a functional RNA-induced silencing complex (RISC). A specific machinery controls miRNA turnover to ensure the silencing release of targeted-genes in given circumstances. The trimming and tailing of miRNAs are fundamental modifications related to their turnover and, hence, to their action. In order to gain a better understanding of these modifications, we analyzed Arabidopsis thaliana small RNA sequencing data from a diversity of mutants, related to miRNA biogenesis, action, and turnover, and from different cellular fractions and immunoprecipitations. Besides confirming the effects of known players in these pathways, we found increased trimming and tailing in miRNA biogenesis mutants. More importantly, our analysis allowed us to reveal the importance of ARGONAUTE 1 (AGO1) loading, slicing activity, and cellular localization in trimming and tailing of miRNAs.

Dose-Dependent AGO1-Mediated Inhibition of the miRNA165/166 Pathway Modulates Stem Cell Maintenance in Arabidopsis Shoot Apical Meristem.

Pluripotent stem cells localized in proliferating growth centers, the meristems, are the origin of life-long organ formation and growth in higher plants. In the shoot apical meristem of Arabidopsis thaliana, the closely related ARGONAUTE proteins AGO1 and ZLL/AGO10 bind miR165/166 species to regulate mRNAs of HD-ZIP III transcription factors that are essential to maintaining stem cells. Several genetic studies showed that AGO1 and ZLL/AGO10 act redundantly to maintain stem cells. By contrast, the reported biochemical data suggested antagonistic functions: AGO1 utilizes miR165/166 to slice HD-ZIP III mRNAs, whereas ZLL/AGO10 promotes degradation of miR165/166 and thus stabilizes HD-ZIP III mRNAs. How these different functions are balanced in stem cell regulation has remained enigmatic. Here, we show that autorepression of AGO1 through miR168-mediated slicing of its own RNA is required to maintain the ability of AGO1 to suppress HD-ZIP III mRNAs. Increased AGO1 expression, either in the miR168a-2 mutant or by transgenic expression, inhibits this ability despite the presence of high levels of miR165/166, effectively uncoupling HD-ZIP III and miR165/166 expression. AGO1 activity can be restored, however, by increasing the levels of chaperones SQN and HSP90, which promote assembly of RNA-induced silencing complex (RISC). This suggests that cellular abundance of SQN and HSP chaperones limits AGO1-mediated RNA interference in shoot meristem stem cell regulation. Localized misexpression of AGO1 indicates that the cells surrounding the shoot meristem primordium play a crucial role in stem cell development. Taken together, our study provides a framework that reconciles biochemical and genetic data, showing that restriction of AGO1 levels by miR168-mediated autorepression is key to RISC homeostasis and the function of AGO1 in stem cell regulation.

Investigating the regulatory roles of the microRNAs and the Argonaute 1-enriched small RNAs in plant metabolism.

The biological roles of small RNAs (sRNAs) in metabolic processes are emerging. However, a systemic study is needed to investigate the wide-spread involvement of the sRNAs in plant metabolism. By using the metabolism-related transcripts retrieved from the public database Plant Metabolic Network, and the publicly available sRNA high-throughput sequencing data, large-scale target identification was performed for microRNAs (miRNAs) and Argonaute 1 (AGO1)-enriched sRNAs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Based on the publicly available degradome sequencing data, 200 miRNA/sRNA-target pairs involving 19 miRNAs, 111 AGO1-enriched sRNAs and 58 target transcripts in Arabidopsis, and 151 pairs involving 62 miRNAs, 33 AGO1-enriched sRNAs and 69 target transcripts in rice were identified. After considering protein-protein interactions for the above identified target genes, a total of 251 pairs involving 21 miRNAs, 120 AGO1-enriched sRNAs and 75 target transcripts exist within the regulatory network of Arabidopsis, and 168 pairs involving 64 miRNAs, 38 AGO1-enriched sRNAs and 80 target transcripts exist in rice. Based on GO (Gene Ontology) term enrichment analysis, the targets within the networks of both plants are enriched in "metabolic process" and "catalytic activity", pointing to the high relevance of the established networks to metabolism. Several functionally conserved subnetworks were identified between the two plant species. Our study provides a basis for studies on metabolism-related sRNAs in plants.

A transcriptome-wide study on the microRNA- and the Argonaute 1-enriched small RNA-mediated regulatory networks involved in plant leaf senescence.

Leaf senescence is an important physiological process during the plant life cycle. However, systemic studies on the impact of microRNAs (miRNAs) on the expression of senescence-associated genes (SAGs) are lacking. Besides, whether other Argonaute 1 (AGO1)-enriched small RNAs (sRNAs) play regulatory roles in leaf senescence remains unclear. In this study, a total of 5,123 and 1,399 AGO1-enriched sRNAs, excluding miRNAs, were identified in Arabidopsis thaliana and rice (Oryza sativa), respectively. After retrieving SAGs from the Leaf Senescence Database, all of the AGO1-enriched sRNAs and the miRBase-registered miRNAs of these two plants were included for target identification. Supported by degradome signatures, 200 regulatory pairs involving 120 AGO1-enriched sRNAs and 40 SAGs, and 266 regulatory pairs involving 64 miRNAs and 42 SAGs were discovered in Arabidopsis. Moreover, 13 genes predicted to interact with some of the above-identified target genes at protein level were validated as regulated by 17 AGO1-enriched sRNAs and ten miRNAs in Arabidopsis. In rice, only one SAG was targeted by three AGO1-enriched sRNAs, and one SAG was targeted by miR395. However, five AGO1-enriched sRNAs were conserved between Arabidopsis and rice. Target genes conserved between the two plants were identified for three of the above five sRNAs, pointing to the conserved roles of these regulatory pairs in leaf senescence or other developmental procedures. Novel targets were discovered for three of the five AGO1-enriched sRNAs in rice, indicating species-specific functions of these sRNA-target pairs. These results could advance our understanding of the sRNA-involved molecular processes modulating leaf senescence.