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MicroRNAs (miRNAs) are short endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level in a broad range of eukaryotic species. In animals, it is estimated that more than 60% of mammalian genes are targets of miRNAs, with miRNAs regulating cellular processes such as differentiation and proliferation. In plants, miRNAs regulate gene expression and play essential roles in diverse biological processes, including growth, development, and stress responses. Arabidopsis mutants with defective miRNA biogenesis are embryo lethal, and abnormal expression of miRNAs can cause severe developmental phenotypes. It is therefore crucial that the homeostasis of miRNAs is tightly regulated. In this review, we summarize the key mechanisms of plant miRNA biogenesis and stabilization. We provide an update on nuclear proteins with functions in miRNA biogenesis and proteins linking miRNA biogenesis to environmental triggers.

P4HA2 has been implicated in various malignant tumors; however, its expression and functional role in colorectal cancer (CRC) remain poorly elucidated. This study aims to investigate the involvement of P4HA2 in CRC metastasis and progression, uncovering the underlying mechanisms. In colorectal cancer (CRC), P4HA2 exhibited overexpression, and elevated levels of P4HA2 expression were associated with an unfavorable prognosis. Functional assays demonstrated P4HA2‘s regulation of cell proliferation, and epithelial–mesenchymal transition (EMT) both in vitro and in vivo. Additionally, the AGO1 expression was correlated with P4HA2, and depletion of AGO1 reversed the proliferation and EMT function induced by P4HA2. Chromatin immunoprecipitation (ChIP) and luciferase assays suggested that the transcription factor SP1 binds to the promoter sequence of P4HA2, activating its expression in CRC. This study unveiled SP1 as a transcriptional regulator of P4HA2 in CRC and AGO1 is a probable target of P4HA2. In conclusion, P4HA2 emerges as a potential prognostic biomarker and promising therapeutic target in colorectal cancer.

MicroRNAs (miRNAs) are short endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level in a broad range of eukaryotic species. In animals, it is estimated that more than 60% of mammalian genes are targets of miRNAs, with miRNAs regulating cellular processes such as differentiation and proliferation. In plants, miRNAs regulate gene expression and play essential roles in diverse biological processes, including growth, development, and stress responses. Arabidopsis mutants with defective miRNA biogenesis are embryo lethal, and abnormal expression of miRNAs can cause severe developmental phenotypes. It is therefore crucial that the homeostasis of miRNAs is tightly regulated. In this review, we summarize the key mechanisms of plant miRNA biogenesis and stabilization. We provide an update on nuclear proteins with functions in miRNA biogenesis and proteins linking miRNA biogenesis to environmental triggers.

Abstract Argonaute (AGO) proteins are highly conserved and widely distributed across various organisms. They mainly associate with small RNAs (sRNAs) and act as integral players of the RNA-induced silencing complex in the RNA interference (RNAi) pathway, regulating gene expression at transcriptional and post-transcriptional levels, thereby mounting diverse fine-tuning functions in a variety of biological processes. Since the discovery and functional characterization of the first AGO in Arabidopsis, our understanding of the functions of AGO proteins has grown rapidly throughout the plant kingdom. AGO1 attracts investigators’ attention because it forms an autoregulatory loop with miR168 and associates with other endogenous sRNAs and cross-kingdom exogenous sRNAs to relay all-round functions. AGO1 associates with endogenous sRNAs that form a complicated regulatory network via targeting a large body of downstream genes involved in growth, development, and stress-induced responses. Host AGO1 may also be exploited by cross-kingdom exogenous sRNAs generated by parasitic organisms to facilitate their colonization via suppressing host defense genes. Moreover, many pathogenic microbes directly target host AGO1 to facilitate their infection via suppression of the host RNAi pathway. Thus, we focus on plant AGO1 and provide an overview of our current understanding of the roles of AGO1 in the coordination of plant disease resistance with growth and development. We also discuss the perspectives in the dissection of the AGO1-mediated signal transduction pathway.

Intellectual disability (ID) and autism spectrum disorders (ASDs) are the most common developmental disorders in humans. Combined, they affect between 3% and 5% of the population. Although high‐throughput genomic methods are rapidly increasing the pool of ASD genes, many cases remain idiopathic. AGO1 is one of the less‐known genes related to ID/ASD. This gene encodes a core member protein of the RNA‐induced silencing complex, which suppresses mRNA expression through cleavage, degradation, and/or translational repression. Generally, patients with defects in the AGO1 gene manifest varying degrees of ID, speech delay, and autistic behaviors. Herein, we used whole‐exome sequencing (WES) to investigate an Iranian family with two affected members one of whom manifested ID and autism and the other showed borderline ID and schizophrenia. WES analysis identified a novel heterozygous truncating variant (NM_012199.5:c.1298G > A, p.Trp433Ter) in the AGO1 gene that co‐segregated with the phenotypes using Sanger sequencing. Moreover, the structural analysis showed that due to this variant, two critical domains (Mid and PIWI) of the AGO1 protein have been lost, which has a detrimental effect on the protein's function and structure. To the best of our knowledge, schizophrenia has not been reported in patients with AGO1 deficiency, which is a novel phenotypic finding that expands the AGO1‐related behavioral disorders. Moreover, this study's findings determined that patients with the same variant in the AGO1 gene may show heterogeneity in manifested phenotypes. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a multifaceted, chronic, progressive autoimmune disease. This study aims to explore the potential benefits of an enhanced drug delivery system utilizing optimized Gelatin Methacryloyl (GelMA) vectors in RA management. We evaluated the levels of miR-1124-3p and AGO1 in RA tissues and cell lines using qPCR, WB, and immunofluorescence. The effects of osthole on inflammatory response and joint morphology were determined by qPCR, H&E staining, and micro-CT. The data showed that miR-1224-3p was downregulated in RA tissues and HUM-iCell-s010RA cells, while the overexpression of miR-1224-3p in HUM-iCell-s010RA cells reduced the expression of IL-6 and IL-1β. Luciferase assay demonstrated that AGO1 was a direct target gene of miR-1224-3p. Additionally, osthole treatment increased miR-1224-3p levels and decreased AGO1 expression. The release data showed that osthole loaded on GelMA was released at a slower rate than free osthole. Further studies in a mouse model of CIA confirmed that osthole-loaded GelMA was more effective in attenuating osteopenia in RA as well as alleviating autoimmune arthritis. These findings suggest that osthole can regulate the miR-1224-3p/AGO1 axis in RASFs cells and has the potential to be developed as a clinical anti-RA drug. GelMA could provide a new approach to long-term RA treatment. [ABSTRACT FROM AUTHOR]

Argonaute (AGO) proteins are highly conserved and widely distributed across various organisms. They mainly associate with small RNAs (sRNAs) and act as integral players of the RNA-induced silencing complex in the RNA interference (RNAi) pathway, regulating gene expression at transcriptional and post-transcriptional levels, thereby mounting diverse fine-tuning functions in a variety of biological processes. Since the discovery and functional characterization of the first AGO in Arabidopsis, our understanding of the functions of AGO proteins has grown rapidly throughout the plant kingdom. AGO1 attracts investigators' attention because it forms an autoregulatory loop with miR168 and associates with other endogenous sRNAs and cross-kingdom exogenous sRNAs to relay all-round functions. AGO1 associates with endogenous sRNAs that form a complicated regulatory network via targeting a large body of downstream genes involved in growth, development, and stress-induced responses. Host AGO1 may also be exploited by cross-kingdom exogenous sRNAs generated by parasitic organisms to facilitate their colonization via suppressing host defense genes. Moreover, many pathogenic microbes directly target host AGO1 to facilitate their infection via suppression of the host RNAi pathway. Thus, we focus on plant AGO1 and provide an overview of our current understanding of the roles of AGO1 in the coordination of plant disease resistance with growth and development. We also discuss the perspectives in the dissection of the AGO1-mediated signal transduction pathway. [ABSTRACT FROM AUTHOR]

Pathogenic AGO1 variants have been associated with neurodevelopmental disorders, including autism spectrum disorder, developmental delay, intellectual disability, and dysmorphic facial appearance. In mammalian models, defects in microRNA (miRNA) biogenesis are associated with congenital heart disease and dilated cardiomyopathy. We describe the case of a patient with partial anomalous pulmonary venous return, hypoplastic left lung, bilateral pulmonary sequestration, and dilated myocardiopathy. We identified a de novo pathogenic variant of AGO1, which encodes an Argonaute protein forming a gene‐silencing complex with microRNAs. The patient was diagnosed with dilated cardiomyopathy with no apparent cause at 3 years of age. She was started on enalapril and carvedilol, and her heart failure was well controlled. We expanded the AGO1‐associated phenotype to include complex congenital cardiovascular anomaly and dilated cardiomyopathy in humans. [ABSTRACT FROM AUTHOR]

Argonaute 1 (AGO1), the principal protein component of microRNA-mediated regulation, plays a key role in plant growth and development. AGO1 physically interacts with the chaperone HSP90, which buffers cryptic genetic variation in plants and animals. We sought to determine whether genetic perturbation of AGO1 in Arabidopsis thaliana would also reveal cryptic genetic variation, and if so, whether AGO1-dependent loci overlap with those dependent on HSP90. To address these questions, we introgressed a hypomorphic mutant allele of AGO1 into a set of mapping lines derived from the commonly used Arabidopsis strains Col-0 and Ler. Although we identified several cases in which AGO1 buffered genetic variation, none of the AGO1-dependent loci overlapped with those buffered by HSP90 for the traits assayed. We focused on 1 buffered locus where AGO1 perturbation uncoupled the traits days to flowering and rosette leaf number, which are otherwise closely correlated. Using a bulk segregant approach, we identified a nonfunctional Ler hua2 mutant allele as the causal AGO1-buffered polymorphism. Introduction of a nonfunctional hua2 allele into a Col-0 ago1 mutant background recapitulated the Lerdependent ago1 phenotype, implying that coupling of these traits involves different molecular players in these closely related strains. Taken together, our findings demonstrate that even though AGO1 and HSP90 buffer genetic variation in the same traits, these robustness regulators interact epistatically with different genetic loci, suggesting that higher-order epistasis is uncommon. [ABSTRACT FROM AUTHOR]

Accumulating evidence has revealed that the dysregulation of circular RNAs (circRNAs) plays crucial roles in the occurrence and progression of cancers. However, the aberrant expression profile and dysfunction of circRNAs in non-small cell lung cancer (NSCLC) have not been fully explored. Herein, we discovered that a circRNA, hsa_circ_0001666 (circ0001666), was highly expressed in NSCLC tissues and cell lines, and it was positively correlated with NSCLC tumor pathological grade and lymph node metastasis. Moreover, Kaplan-Meier survival analysis implied that NSCLC patients with high circ0001666 expression were negatively correlated with favorable survival. Functionally, circ0001666 could promote migration and invasion of NSCLC cells in vitro and in vivo. Mechanistically, circ0001666 could act as a sponge to miR-1184/miR-548I and upregulate the expression of AGO1, thereby promoting the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway in NSCLC cells. Collectively, these findings demonstrated that circ0001666 could serve as an oncogene to promote the migration and invasion of NSCLC via a novel miR-1184/miR-548I/AGO1 axis, which might be a promising therapeutic target for NSCLC treatment.

RNA silencing negatively regulates gene expression at the transcriptional and post-transcriptional levels through DNA methylation, histone modification, mRNA cleavage, and translational inhibition. Small interfering RNAs (siRNAs) of 21 to 24 nucleotides are processed from double-stranded RNAs by Dicer-like enzymes and play essential roles in RNA silencing in plants. Here, we demonstrated that ALTERED MERISTEM PROGRAM1 (AMP1) and its putative paralog LIKE AMP1 (LAMP1) impair RNA silencing by repressing the biogenesis of a subset of inverted repeat (IR)-derived siRNAs in Arabidopsis (Arabidopsis thaliana). AMP1 and LAMP1 inhibit Pol II-dependent IR gene transcription by suppressing ARGONAUTE 1 (AGO1) protein levels. Genetic analysis indicates that AMP1 acts upstream of RNA polymerase IV subunit 1 (NRPD1), RNA-dependent RNA polymerase 2 (RDR2), and Dicer-Like 4 (DCL4), which are required for IR-induced RNA silencing. We also show that AMP1 and LAMP1 inhibit siRNA-mediated silencing in a different mechanism from AGO4 and DCL3. Together, these results reveal two previously unknown players in siRNA biogenesis from IRs - AGO1, which promotes IR transcription, and AMP1, which inhibits IR transcription indirectly through the repression of AGO1 expression., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Rheumatoid arthritis (RA) is a multifaceted, chronic, progressive autoimmune disease. This study aims to explore the potential benefits of an enhanced drug delivery system utilizing optimized Gelatin Methacryloyl (GelMA) vectors in RA management. We evaluated the levels of miR-1124-3p and AGO1 in RA tissues and cell lines using qPCR, WB, and immunofluorescence. The effects of osthole on inflammatory response and joint morphology were determined by qPCR, H&E staining, and micro-CT. The data showed that miR-1224-3p was downregulated in RA tissues and HUM-iCell-s010RA cells, while the overexpression of miR-1224-3p in HUM-iCell-s010RA cells reduced the expression of IL-6 and IL-1β. Luciferase assay demonstrated that AGO1 was a direct target gene of miR-1224-3p. Additionally, osthole treatment increased miR-1224-3p levels and decreased AGO1 expression. The release data showed that osthole loaded on GelMA was released at a slower rate than free osthole. Further studies in a mouse model of CIA confirmed that osthole-loaded GelMA was more effective in attenuating osteopenia in RA as well as alleviating autoimmune arthritis. These findings suggest that osthole can regulate the miR-1224-3p/AGO1 axis in RASFs cells and has the potential to be developed as a clinical anti-RA drug. GelMA could provide a new approach to long-term RA treatment.

To study the morphogenesis mechanism of leaf primordium differentiation, we used Liriodendron tulipifera to clone 2 001 bp upstream region of LtAGO1 CDS as the promoter by RT-PCR and RACE technology and predicted its function, and we also used real-time PCR to investigate expression patterns in Liriodendron, obtained the transgenic Arabidopsis thaliana of ProAGO1∷GUS by resistance screening and DNA dentification, and then monitored phenotype and GUS histochemical staining. The results were as follows:（1） The LtAGO1 gene included an open reading frame for 3 300 bp, encoding 1 100 amino acid, the molecular weight was 122.14 kD and theoretical isoelectric point（pI） was 9.36.（2）Amino acid sequence analysis showed that LtAGO1 consisted of Gly-rich-AGO1 and Piwi conserved domains of AGO family. Phylogenetic trees revealed that LtAGO1 was closed to Cinnamomum micranthum（RWR84608.1） in evolutional relationship.（3）The specific tissue expression analysis demonstrated that the expression order was stamen>floral bud>petal>calyx>leaf>pistil>leaf bud>stem among tissues, the expression order was leaf bud sprouting stage >young leaf stage>senescence stage >mature stage among stages, it was highly expressed in the leaf margin of Liriodendron L., and LtAGO1 gene expression in leaf tooth sinus was higher than that in leaf tooth tip of Liriodendron tulipifera.（4）The transgenic strains leaf polarity of the medio-lateral and proximo-distal axis were absent with serrated leaf margin and double petal flower. It was found that GUS staining was stably detected at the tip of leaf bud of transgenic seedlings, and higher GUS activity was observed at newly differentiated petioles. ProAGO1 promoter drove GUS gene to accumulate specifically in the vascular bundle of Arabidopsis thaliana leaf, flower, pod and stem, and GUS activity intensity order was leaf tip bud> flower>vascular bundle among tissues, which was in accordance with the real-time PCR results. Therefore, the LtAGO1 gene is predominantly expressed in apical meristem and is regulated by various pathways during the development of leaf and flower. The results provide a theoretical basis for further functional research of the LtAGO1 gene in Liriodendron tulipifera and regulation mechanism of leaf shape development. [ABSTRACT FROM AUTHOR]

AGO1, as one of the rare genes in neurodevelopmental disorders, is involved in the microRNA‐induced silencing complex. Here, we describe the clinical and genetic features of 18 individuals with de novo AGO1 variants: four new and 14 previously reported. Three variants are identified: two in‐frame deletion variants and one missense variant. The spectrum of AGO1‐related disorders included global development delay (GDD), intellectual disability (ID) with or without epilepsy, autism spectrum disorder, hypotonia and dysmorphisms. Focal seizures are the most common type of seizure, occasionally with atypical absence. Mild deafness may be a new phenotype of AGO1‐releated disease. Gly199Ser may be a hot‐spot variant of AGO1 with the same phenotype: GDD/ID, intractable epilepsy, remarkably with Rolandic discharges, and even reaching electrical status epilepticus during sleep. [ABSTRACT FROM AUTHOR]

Communication between interacting organisms via bioactive molecules is widespread in nature and plays key roles in diverse biological processes. Small RNAs (sRNAs) can travel between host plants and filamentous pathogens to trigger transkingdom RNA interference (RNAi) in recipient cells and modulate plant defense and pathogen virulence. However, how fungal pathogens counteract transkingdom antifungal RNAi has rarely been reported. Here we show that a secretory protein VdSSR1 (secretory silencing repressor 1) from Verticillium dahliae, a soil-borne phytopathogenic fungus that causes wilt diseases in a wide range of plant hosts, is required for fungal virulence in plants. VdSSR1 can translocate to plant nucleus and serve as a general suppressor of sRNA nucleocytoplasmic shuttling. We further reveal that VdSSR1 sequesters ALY family proteins, adaptors of the TREX complex, to interfere with nuclear export of the AGO1–microRNA (AGO1–miRNA) complex, leading to a great attenuation in cytoplasmic AGO1 protein and sRNA levels. With this mechanism, V. dahliae can suppress the accumulation of mobile plant miRNAs in fungal cells and succedent transkingdom silencing of virulence genes, thereby increasing its virulence in plants. Our findings reveal a mechanism by which phytopathogenic fungi antagonize antifungal RNAidependent plant immunity and expand the understanding on the complex interaction between host and filamentous pathogens. [ABSTRACT FROM AUTHOR]

P4HA2 has been implicated in various malignant tumors; however, its expression and functional role in colorectal cancer (CRC) remain poorly elucidated. This study aims to investigate the involvement of P4HA2 in CRC metastasis and progression, uncovering the underlying mechanisms. In colorectal cancer (CRC), P4HA2 exhibited overexpression, and elevated levels of P4HA2 expression were associated with an unfavorable prognosis. Functional assays demonstrated P4HA2's regulation of cell proliferation, and epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Additionally, the AGO1 expression was correlated with P4HA2, and depletion of AGO1 reversed the proliferation and EMT function induced by P4HA2. Chromatin immunoprecipitation (ChIP) and luciferase assays suggested that the transcription factor SP1 binds to the promoter sequence of P4HA2, activating its expression in CRC. This study unveiled SP1 as a transcriptional regulator of P4HA2 in CRC and AGO1 is a probable target of P4HA2. In conclusion, P4HA2 emerges as a potential prognostic biomarker and promising therapeutic target in colorectal cancer.

Abstract MicroRNA are small non-coding RNAs that post-transcriptionally regulate the expression levels of messenger RNAs. MicroRNA regulation activity depends on the recognition of binding sites located on mRNA molecules. ComiR is a web tool realized to predict the targets of a set of microRNAs, starting from their expression profile. ComiR was trained with the information regarding binding sites in the 3’utr region, by using a reliable dataset containing the targets of endogenously expressed microRNA in D. melanogaster S2 cells. This dataset was obtained by comparing the results from two different experimental approaches, i.e., inhibition, and immunoprecipitation of the AGO1 protein--a component of the microRNA induced silencing complex. In this work, we tested whether including coding region binding sites in ComiR algorithm improves the performance of the tool in predicting microRNA targets. We focused the analysis on the D. melanogaster species and updated the ComiR underlying database with the currently available releases of mRNA and microRNA sequences. As a result, we find that ComiR algorithm trained with the information related to the coding regions is more efficient in predicting the microRNA targets, with respect to the algorithm trained with 3’utr information. On the other hand, we show that 3’utr based predictions can be seen as complementary to the coding region based predictions, which suggests that both predictions, from 3’utr and coding regions, should be considered in comprehensive analysis. Furthermore, we observed that the lists of targets obtained by analyzing data from one experimental approach only, that is, inhibition or immunoprecipitation of AGO1, are not reliable enough to test the performance of our microRNA target prediction algorithm. Further analysis will be conducted to investigate the effectiveness of the tool with data from other species, provided that validated datasets, as obtained from the comparison of RISC proteins inhibition and immunoprecipitation experiments, will be available for the same samples. Finally, we propose to upgrade the existing ComiR web-tool by including the coding region based trained model, available together with the 3’utr based one.

Translational repression is a conserved mechanism in microRNA (miRNA)‐guided gene silencing. In Arabidopsis, ARGONAUTE1 (AGO1), the major miRNA effector, localizes in the cytoplasm for mRNA cleavage and at the endoplasmic reticulum (ER) for translational repression of target genes. However, the mechanism underlying miRNA‐mediated translational repression is poorly understood. In particular, how the subcellular partitioning of AGO1 is regulated is largely unexplored. Here, we show that the plant hormone brassinosteroids (BRs) inhibit miRNA‐mediated translational repression by negatively regulating the distribution of AGO1 at the ER in Arabidopsis thaliana. We show that the protein levels rather than the transcript levels of miRNA target genes were reduced in BR‐deficient mutants but increased under BR treatments. The localization of AGO1 at the ER was significantly decreased under BR treatments while it was increased in the BR‐deficient mutants. Moreover, ROTUNDIFOLIA3 (ROT3), an enzyme involved in BR biosynthesis, co‐localizes with AGO1 at the ER and interacts with AGO1 in a GW motif‐dependent manner. Complementation analysis showed that the AGO1–ROT3 interaction is necessary for the function of ROT3. Our findings provide new clues to understand how miRNA‐mediated gene silencing is regulated by plant endogenous hormones. [ABSTRACT FROM AUTHOR]

Argonaute (AGO) proteins are evolutionarily conserved RNA-binding proteins that control gene expression through the small RNAs they interact with. Whether AGOs have regulatory roles independent of RNAs, however, is unknown. Here, we show that AGO1 controls cell fate decisions through facilitating protein folding. We found that in mouse embryonic stem cells (mESCs), while AGO2 facilitates differentiation via the microRNA (miRNA) pathway, AGO1 controls stemness independently of its binding to small RNAs. We determined that AGO1 specifically interacts with HOP, a co-chaperone for the HSP70 and HSP90 chaperones, and enhances the folding of a set of HOP client proteins with intrinsically disordered regions. This AGO1-mediated facilitation of protein folding is important for maintaining stemness in mESCs. Our results demonstrate divergent functions between AGO1 and AGO2 in controlling cellular states and identify an RNA-independent function of AGO1 in controlling gene expression and cell fate decisions. [Display omitted] • Opposite functions of AGO1 and AGO2 in controlling self-renewal and differentiation • AGO1 promotes stemness independently of binding to small RNAs • AGO1 controls protein folding through interacting with HOP in stem cells Liu et al. show that AGO1 and AGO2 have distinct functions in controlling stem cell self-renewal and differentiation. While AGO2 facilitates differentiation through the miRNA pathway, AGO1 promotes stemness in a small-RNA-independent fashion by controlling protein folding. [ABSTRACT FROM AUTHOR]

Drosophila model is intensively studied for the development of cancer. The diminutive (dMyc), a homolog of the human MYC gene, is responsible for cell-apoptosis and its upregulation is responsible for determining the fate of cancerous growth in humans and Drosophila model. This work implores the requirement of dMyc and its expression as one of the major regulator of cancer with other proteins and repression of dMyc mRNA in Drosophila S2 cells. Here we report protein complex of Argonaute 1 (AGO1), Bag of marbles (Bam), and Brain tumor (Brat) proteins and not the individual factor of this complex repression of dMyc mRNA in Drosophila Schneider 2 cells and promote differentiation in cystoblast of Drosophila ovary. These results exhibit the significant role of this complex, including master differentiation factor Bam with other various differentiation factor Brat and microRNA pathway component AGO1, which may negatively regulate dMyc mRNA and so the dMyc protein. [ABSTRACT FROM AUTHOR]

Previous study has shown that Hibiscus sulfite oxidase (SO) interacts with Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) and triggers sulfur enhanced defense (SED). In this study, we show the interaction of Arabidopsis SO (AtSO) and Turnip crinkle virus (TCV) CP in Arabidopsis thaliana plants. We identified the binding sites of TCV CP (W274) and AtSO (D223) using bioinformatics and confirmed it experimentally. Mutation of binding site W274 to A274 in TCV CP resulted in failure of TCV infection. TCV accumulation in SO over-expression (SO_OE) plants was lower than that in wild-type (WT) and SO knock-out (SO_KO) plants at 7 dpi but reached a level similar to that of WT and SO_KO plants at 10 dpi. AtSO competed with Argonaute 1 (AGO1) for TCV CP binding in vitro. AtSO may serve as an anti-viral factor through sequestering TCV CP for binding with AGO1 and confers virus resistance. [ABSTRACT FROM AUTHOR]

Recent studies have established important roles of de novo mutations (DNMs) in autism spectrum disorders (ASDs). Here, we analyze DNMs in 262 ASD probands of Japanese origin and confirm the “de novo paradigm” of ASDs across ethnicities. Based on this consistency, we combine the lists of damaging DNMs in our and published ASD cohorts (total number of trios, 4,244) and perform integrative bioinformatics analyses. Besides replicating the findings of previous studies, our analyses highlight ATP-binding genes and fetal cerebellar/striatal circuits. Analysis of individual genes identified 61 genes enriched for damaging DNMs, including ten genes for which our dataset now contributes to statistical significance. Screening of compounds altering the expression of genes hit by damaging DNMs reveals a global downregulating effect of valproic acid, a known risk factor for ASDs, whereas cardiac glycosides upregulate these genes. Collectively, our integrative approach provides deeper biological and potential medical insights into ASDs.

Background: Metabolic disorders such as obesity and diabetes mellitus can cause dysfunction of endothelial cells (ECs) and vascular rarefaction in adipose tissues. However, the modulatory role of ECs in adipose tissue function is not fully understood. Other than vascular endothelial growth factor-vascular endothelial growth factor receptor-mediated angiogenic signaling, little is known about the EC-derived signals in adipose tissue regulation. We previously identified Argonaute 1 (AGO1; a key component of microRNA-induced silencing complex) as a crucial regulator in hypoxia-induced angiogenesis. In this study, we intend to determine the AGO1-mediated EC transcriptome, the functional importance of AGO1-regulated endothelial function in vivo, and the relevance to adipose tissue function and obesity.Methods: We generated and subjected mice with EC-AGO1 deletion (EC-AGO1-knockout [KO]) and their wild-type littermates to a fast food-mimicking, high-fat high-sucrose diet and profiled the metabolic phenotypes. We used crosslinking immunoprecipitation- and RNA-sequencing to identify the AGO1-mediated mechanisms underlying the observed metabolic phenotype of EC-AGO1-KO. We further leveraged cell cultures and mouse models to validate the functional importance of the identified molecular pathway, for which the translational relevance was explored using human endothelium isolated from healthy donors and donors with obesity/type 2 diabetes mellitus.Results: We identified an antiobesity phenotype of EC-AGO1-KO, evident by lower body weight and body fat, improved insulin sensitivity, and enhanced energy expenditure. At the organ level, we observed the most significant phenotype in the subcutaneous and brown adipose tissues of KO mice, with greater vascularity and enhanced browning and thermogenesis. Mechanistically, EC-AGO1 suppression results in inhibition of thrombospondin-1 (THBS1/TSP1), an antiangiogenic and proinflammatory cytokine that promotes insulin resistance. In EC-AGO1-KO mice, overexpression of TSP1 substantially attenuated the beneficial phenotype. In human endothelium isolated from donors with obesity or type 2 diabetes mellitus, AGO1 and THBS1 are expressed at higher levels than the healthy controls, supporting a pathological role of this pathway.Conclusions: Our study suggests a novel mechanism by which ECs, through the AGO1-TSP1 pathway, control vascularization and function of adipose tissues, insulin sensitivity, and whole-body metabolic state. [ABSTRACT FROM AUTHOR]

The steroid hormone ecdysone is the central regulator of insect metamorphosis, during which a growing, immature larva is remodeled, through pupal stages, to a reproductive adult. However, the underlying mechanisms of ecdysone‐mediated metamorphosis remain to be fully elucidated. Here, we identified metamorphosis‐associated microRNAs (miRNAs) and their potential targets by cross‐linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 protein in Drosophila. Interestingly, miR‐8‐3p targeted five Vha genes encoding distinct subunits of vacuolar H+‐ATPase (V‐ATPase), which has a vital role in the organellar acidification. The expression of ecdysone‐responsive miR‐8‐3p is normally downregulated during Drosophila metamorphosis, but temporary overexpression of miR‐8‐3p in the whole body at the end of larval development led to defects in metamorphosis and survival, hallmarks of aberrant ecdysone signaling. In addition, miR‐8‐3p was expressed in the prothoracic gland (PG), which produces and releases ecdysone in response to prothoracicotropic hormone (PTTH). Notably, overexpression of miR‐8‐3p or knockdown of its Vha targets in the PG resulted in larger than normal, ecdysone‐deficient larvae that failed to develop into the pupal stage but could be rescued by ecdysone feeding. Moreover, these animals showed defective PTTH signaling with a concomitant decrease in the expression of ecdysone biosynthetic genes. We also demonstrated that the regulatory network between the conserved miR‐8‐3p/miR‐200 family and V‐ATPase was functional in human cells. Consequently, our data indicate that the coordinated regulation of V‐ATPase subunits by miR‐8‐3p is involved in Drosophila metamorphosis by controlling the ecdysone biosynthesis. [ABSTRACT FROM AUTHOR]

The transcriptional and posttranscriptional AGO-mediated control of gene expression may play important roles during male monocot gametophyte development. In this report, we demonstrated dynamic changes in the spatiotemporal distribution of AGO1 and AGO4, which are key proteins of the RNA-induced silencing complex (RISC) in Hyacinthus orientalis male gametophyte development. During maturation of the bicellular pollen grains and in vitro pollen tube growth, the pattern of AGO1 localization was correlated with previously observed transcriptional activity of the cells. During the period of high transcriptional activity, AGO1 is associated with chromatin while the clustered distribution of AGO1 in the interchromatin areas is accompanied by condensation of chromatin and the gradual transcriptional silencing of both cells in mature, dehydrated pollen. During pollen tube growth and the restarting of RNA synthesis in the vegetative nucleus, AGO1 is dispersed in the chromatin. Additionally, the gradual increase in the cytoplasmic pool of AGO1 in the elongating pollen tube indicates the activation of the posttranscriptional gene silencing (PTGS) pathway. During pollen tube growth in the generative cell and in the sperm cells, AGO1 is present mainly in the areas between highly condensed chromatin clusters. Changes in the distribution of AGO4 that indicated the possibility of spatiotemporal organization in the RNA-directed DNA methylation (RdDM) process (cytoplasmic and nuclear steps) were also observed during hyacinth male gametophyte development. Based on our findings, we propose that in the germinating pollen tube, the cytoplasmic assembly of AGO4/siRNA takes place and that the mature complexes could be transported to the nucleus to carry out their function during the next steps of pollen tube growth. [ABSTRACT FROM AUTHOR]

Ostrinia furnacalis (Lepidoptera: Pyralidae) is one of the most destructive agricultural pests in Asia. Traditional pest‐management methods include sex pheromone capture, transgenic crops that produce Bacillus thuringiensis toxin, and pesticides. Although these strategies control pest populations effectively, they also cause negative side effects, including dramatically increased pesticide resistance, severe pollution, and hazards for human health. Recently developed genome editing tools provide new prospects for pest management and have been successfully used in several species. However, few examples have been reported in the agricultural pest O. furnacalis due to a lack in genomic information. In this report, we identified only one transcript of O. furnacalis Argonaute 1 (OfAgo1) gene from the genome and cloned the open reading frame. OfAgo1 presented the maximum expression at the embryo stage or in the fat body during the larval stages. To understand its function, an OfAgo1 mutant was constructed using the Clustered Regularly Interspaced Short Palindromic Repeat/RNA‐guided Cas9 nuclease (CRISPR/Cas9). Mutagenesis of OfAgo1 disrupted cuticle pigmentation by down‐regulating micro RNAs and pigmentation‐related genes. This is the first report for the cloning and functional analysis of OfAgo1, revealing a role of OfAgo1 in cuticle pigmentation. The current report also established a CRISPR/Cas9 system in O. furnacalis, providing a new insight for pest management. [ABSTRACT FROM AUTHOR]

RNA silencing is a sequence specific post-transcriptional mechanism regulating important biological processes including antiviral defense in plants. Argonaute (AGO) proteins, the catalytic subunits of the silencing complexes, are loaded with small RNAs to execute the sequence specific RNA cleavage or translational inhibition. Plants encode several AGO proteins and a few of them, especially AGO1 and AGO2, have been shown to be required for antiviral silencing. Previously, we have shown that the P1 protein of the sweet potato mild mottle virus (SPMMV) suppresses the primary RNA silencing response by inhibiting AGO1. To analyze the role of AGO2 in antiviral defense against the SPMMV, we performed a comparative study using a wild type and ago2−/− mutant Nicotiana benthamiana. Here we show that the AGO2 of N. benthamiana attenuates the symptoms of SPMMV infection. Upon SPMMV infection the levels of AGO2 mRNA and protein are greatly increased. Moreover, we found that AGO2 proteins are loaded with SPMMV derived viral small RNAs as well as with miRNAs. Our results indicate that AGO2 protein takes over the place of AGO1 to confer antiviral silencing. Finally, we provide a plausible explanation for the AGO2 mediated recovery of an SPMMV-infected sweet potato.

[Display omitted] • The rate of translation inversely regulates the efficiency of stop codon readthrough (SCR) • Global reduction of translation rate enhances the efficiency of SCR. • Local reduction of translation rate by rare codons enhances the efficiency of SCR. During translation, a stop codon on the mRNA signals the ribosomes to terminate the process. In certain mRNAs, the termination fails due to the recoding of the canonical stop codon, and ribosomes continue translation to generate C-terminally extended protein. This process, termed stop codon readthrough (SCR), regulates several cellular functions. SCR is driven by elements/factors that act immediately downstream of the stop codon. Here, we have analysed the process of SCR using a simple mathematical model to investigate how the kinetics of translating ribosomes influences the efficiency of SCR. Surprisingly, the analysis revealed that the rate of translation inversely regulates the efficiency of SCR. We tested this prediction experimentally in mammalian AGO1 and MTCH2 mRNAs. Reduction in translation either globally by harringtonine or locally by rare codons caused an increase in the efficiency of SCR. Thus, our study has revealed a hitherto unknown mode of regulation of SCR. [ABSTRACT FROM AUTHOR]

To counteract host antiviral RNA silencing, plant viruses encode numerous viral suppressors of RNA silencing (VSRs). P0 proteins have been identified as VSRs in many poleroviruses. However, their suppressor function has not been fully characterized. Here, we investigated the function of P0 from pea mild chlorosis virus (PMCV) in the suppression of local and systemic RNA silencing via green fluorescent protein (GFP) co-infiltration assays in wild-type and GFP-transgenic Nicotiana benthamiana (line 16c). Amino acid deletion analysis showed that N-terminal residues Asn 2 and Val 3, but not the C-terminus residues from 230–270 aa, were necessary for PMCV P0 (P0PM) VSR activity. P0PM acted as an F-box protein, and triple LPP mutation (62LPxx79P) at the F-box-like motif abolished its VSR activity. In addition, P0PM failed to interact with S-phase kinase-associated protein 1 (SKP1), which was consistent with previous findings of P0 from potato leafroll virus. These data further support the notion that VSR activity of P0 is independent of P0–SKP1 interaction. Furthermore, we examined the effect of P0PM on ARGONAUTE1 (AGO1) protein stability, and co-expression analysis showed that P0PM triggered AGO1 degradation. Taken together, our findings suggest that P0PM promotes degradation of AGO1 to suppress RNA silencing independent of SKP1 interaction.

The steroid hormone ecdysone has a central role in the developmental transitions of insects through its control of responsive protein-coding and microRNA (miRNA) gene expression. However, the complete regulatory network controlling the expression of these genes remains to be elucidated. In this study, we performed cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 (Ago1) protein, the core effector of the miRNA pathway, in Drosophila S2 cells. We found that regulatory interactions between miRNAs and their cognate targets were substantially altered by Ago1 in response to ecdysone signaling. Additionally, during the larva-to-adult metamorphosis, miR-252-5p was up-regulated via the canonical ecdysone-signaling pathway. Moreover, we provide evidence that miR-252-5p targets Abelson interacting protein (Abi) to decrease the protein levels of cyclins A and B, controlling the cell cycle. Overall, our data suggest a potential role for the ecdysone/miR-252-5p/Abi regulatory axis partly in cell-cycle control during metamorphosis in Drosophila. [ABSTRACT FROM AUTHOR]

The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development, and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 represses its targets through RNA-supported interaction with TNRC6/GW182, a core component of the miRNA-induced silencing complex (miRISC). We demonstrate that AGO2, TRIM71, and UPF1 each recruit TNRC6 to specific sets of transcripts to silence them. As cellular TNRC6 levels are limiting, competition occurs among the silencing pathways, such that the loss of AGO proteins or of AGO binding to TNRC6 enhances the activities of the other pathways. We conclude that a miRNA-like silencing activity is shared among different mRNA silencing pathways and that the use of TNRC6 as a central hub provides a means to integrate their activities. [Display omitted] • sgRNA screens show enhanced TRIM71-mediated RNA silencing upon miRNA pathway disruption • The miRISC component GW182/TNRC6 acts in AGO-, TRIM71-, and UPF1-mediated RNA silencing • AGO2, TRIM71, and UPF1 recruit GW182/TNRC6A to specific sets of target transcripts • AGO proteins antagonize TRIM71 and UPF1 activity by sequestering GW182/TNRC6 Welte et al. demonstrate an unexpected crosstalk between major cellular RNA-silencing pathways. This is driven by a common silencing factor (TNRC6). One RNA-silencing pathway—the miRNA pathway—can control the activity of other pathways by restricting their access to TNRC6. [ABSTRACT FROM AUTHOR]

Recent researches have uncovered that long non-coding RNAs (lncRNAs) are closely correlated with the development of different diseases, while biological functions and hidden molecular mechanisms of antisense lncRNAs in oesophageal squamous cell carcinoma (OSCC) remain unclear. Here, we identified upregulation of LINC01116 in RNA sequencing data, online database, and in OSCC and intraepithelial neoplasia (IEN) specimens. Functionally, LINC01116 facilitates OSCC advancement and metastasis in vitro and vivo. Mechanistically, elevated expression of LINC01116 in OSCC cells other than tumor stroma and cytoplasmic enables it to activate AGO1 expression via complementary binding with AGO1 mRNA to facilitate EMT process of OSCC. • The upregulation of LINC01116 in OSCC and IEN tissues and its relevance with cliniopathological parameters was identified. • The cytoplasmic expression of LIN01116 other than stroma promotes EMT process of OSCC cells. • Repression of LINC01116 impeded AGO1-meadiated EMT and interfering AGO1 could partially rescue the impact on invasion and EMT of OSCC. [ABSTRACT FROM AUTHOR]

Bladder cancer still requires improvements in diagnosis and prognosis, because many of the cases will recur and/or metastasize with bad outcomes. Despite ongoing research on bladder biomarkers, the clinicopathological impact and diagnostic function of miRNA maturation regulators Drosha and Argonaute proteins AGO1 and AGO2 in urothelial bladder carcinoma remain unclear. Therefore, we conducted immunohistochemical investigations of a tissue microarray composed of 112 urothelial bladder carcinomas from therapy-naïve patients who underwent radical cystectomy or transurethral resection and compared the staining signal with adjacent normal bladder tissue. The correlations of protein expression of Drosha, AGO1 and AGO2 with sex, age, tumor stage, histological grading and overall survival were evaluated in order to identify their diagnostic and prognostic potential in urothelial cancer. Our results show an upregulation of AGO1, AGO2 and Drosha in non-muscle-invasive bladder carcinomas, while there was increased protein expression of only AGO2 in muscle-invasive bladder carcinomas. Moreover, we were able to differentiate between non-muscle-invasive and muscle-invasive bladder carcinoma according to AGO1 and Drosha expression. Finally, despite Drosha being a discriminating factor that can predict the probability of overall survival in the Kaplan–Meier analysis, AGO1 turned out to be independent of all clinicopathological parameters according to Cox regression. In conclusion, we assumed that the miRNA processing factors have clinical relevance as potential diagnostic and prognostic tools for bladder cancer.

Eukaryotic Argonaute proteins play primary roles in miRNA and siRNA pathways that are essential for numerous developmental and biological processes. However, the functional roles of the four ZmAGO1 genes have not yet been characterized in maize ( Zea mays L.). In the present study, ZmAGO1a was identified from four putative ZmAGO1 genes for further characterization. Complementation of the Arabidopsis ago1-27 mutant with ZmAGO1a indicated that constitutive overexpression of ZmAGO1a could restore the smaller rosette, serrated leaves, later flowering and maturation, lower seed set, and darker green leaves at late stages of the mutant to the wild-type phenotype. The expression profiles of ZmAGO1a under five different abiotic stresses indicated that ZmAGO1a shares expression patterns similar to those of Argonaute genes in rice, Arabidopsis, and wheat. Further, variation in ZmAGO1a alleles among diverse maize germplasm that resulted in several amino acid changes revealed genetic diversity at this locus. The present data suggest that ZmAGO1a might be an important AGO1 ortholog in maize. The results presented provide further insight into the function of ZmAGO1a. [ABSTRACT FROM AUTHOR]

Post-translational modifications (PTMs) of proteins are essential to increase the functional diversity of the proteome. By adding chemical groups to proteins, or degrading entire proteins by phosphorylation, glycosylation, ubiquitination, neddylation, acetylation, lipidation, and proteolysis, the complexity of the proteome increases, and this then influences most biological processes. Although small RNAs are crucial regulatory elements for gene expression in most eukaryotes, PTMs of small RNA microprocessor and RNA silencing components have not been extensively investigated in plants. To date, several studies have shown that the proteolytic regulation of AGOs is important for host-pathogen interactions. DRB4 is regulated by the ubiquitin-proteasome system, and the degradation of HYL1 is modulated by a de-etiolation repressor, COP1, and an unknown cytoplasmic protease. Here, we discuss current findings on the PTMs of microprocessor and RNA silencing components in plants. [ABSTRACT FROM AUTHOR]

Foxtail millet (Setaria italica (L.) P. Beauv), which belongs to the Panicoideae tribe of the Poaceae, is an important grain crop widely grown in Northern China and India. It is currently developing into a novel model species for functional genomics of the Panicoideae as a result of its fully available reference genome sequence, small diploid genome (2n=18, ~510 Mb), short life cycle, small stature and prolific seed production. Argonaute 1 (AGO1), belonging to the argonaute (AGO) protein family, recruits small RNAs and regulates plant growth and development. Here, we characterized an AGO1 mutant (siago1b) in foxtail millet, which was induced by ethyl methanesulfonate treatment. The mutant exhibited pleiotropic developmental defects, including dwarfing stem, narrow and rolled leaves, smaller panicles and lower rates of seed setting. Map-based cloning analysis demonstrated that these phenotypic variations were attributed to a C-A transversion, and a 7-bp deletion in the C-terminus of the SiAGO1b gene in siago1b. Yeast two-hybrid assays and BiFC experiments revealed that the mutated region was an essential functional motif for the interaction between SiAGO1b and SiHYL1. Furthermore, 1598 differentially expressed genes were detected via RNAseq- based comparison of SiAGO1b and wild-type plants, which revealed that SiAGO1b mutation influenced multiple biological processes, including energy metabolism, cell growth, programmed death and abiotic stress responses in foxtail millet. This study may provide a better understanding of the mechanisms by which SiAGO1b regulates the growth and development of crops. [ABSTRACT FROM AUTHOR]

RNA silencing refers to a conserved RNA-directed gene regulatory mechanism in a wide range of eukaryotes. It plays an important role in many processes including growth, development, genome stability, and antiviral defense in the plants. Geminivirus encoded AC2 is identified as an RNA silencing suppressor protein, however, the mechanism of action has not been characterized. In this paper, we elucidate another mechanism of AC2-mediated suppression activity of Mungbean Yellow Mosaic India Virus (MYMIV). The AC2 protein, unlike many other suppressors, does not bind to siRNA or dsRNA species and its suppression activity is mediated through interaction with key components of the RNA silencing pathway, viz., RDR6 and AGO1. AC2 interaction inhibits the RDR6 activity, an essential component of siRNA and tasi-RNA biogenesis and AGO1, the major slicing factor of RISC. Thus the study identifies dual sites of MYMIV-AC2 interference and probably accounts for its strong RNA silencing suppression activity. [ABSTRACT FROM AUTHOR]

The processing of miRNA from its precursors is a precisely regulated process and after biogenesis, the miRNAs are amenable to different kinds of modifications by the addition or deletion of nucleotides at the terminal ends. However, the mechanism and functions of such modifications are not well studied in plants. In this study, we have specifically analysed the terminal end non-templated miRNA modifications, using NGS data of rice, tomato and Arabidopsis small RNA transcriptomes from different tissues and physiological conditions. Our analysis reveals template independent terminal end modifications in the mature as well as passenger strands of the miRNA duplex. Interestingly, it is also observed that miRNA sequences terminating with a cytosine (C) at the 3′ end undergo a higher percentage of 5′ end modifications. The terminal end modifications did not correlate with the miRNA abundances and are independent of tissue types, physiological conditions and plant species. Our analysis indicates that the addition of nucleotides at miRNA ends is not influenced by the absence of RNA dependent RNA polymerase 6. Moreover the terminal end modified miRNAs are also observed amongst AGO1 bound small RNAs and have potential to alter target, indicating its important functional role in repression of gene expression. [ABSTRACT FROM AUTHOR]

Profiling small RNAs in soybean Williams 82 ( rsv ), susceptible to Soybean mosaic virus (SMV, the genus Potyvirus , family Potyviridae ) strains G2 and G7, and soybean PI96983 ( Rsv1 ), resistant to G2 but susceptible to G7, identified the microRNA miR168 that was highly overexpressed only in G7-infected PI96983 showing a lethal systemic hypersensitive response (LSHR). Overexpression of miR168 was in parallel with the high-level expression of AGO1 mRNA, high-level accumulation of miR168-mediated AGO1 mRNA cleavage products but with severely repressed AGO1 protein. In contrast, AGO1 mRNA, degradation products and protein remained without significant changes in G2- and G7-infected Williams 82. Moreover, knock-down of SGS3 , an essential component in RNA silencing, suppressed AGO1 siRNA, partially recovered repressed AGO1 protein, and alleviated LSHR severity in G7-infected Rsv1 soybean. These results suggest that both miRNA and siRNA pathways are involved in G7 infection of Rsv1 soybean, and LSHR is associated with breakdown of AGO1 homeostasis. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNA) are small (21-24 nt) non-coding RNAs involved in many biological processes in both plants and animals. The biogenesis of plant miRNAs starts with the transcription of MIRNA (MIR ) genes by RNA polymerase II; then, the primary miRNA transcripts are cleaved by Dicer-like proteins into mature miRNAs, which are then loaded into Argonaute (AGO) proteins to form the effector complex, the miRNA-induced silencing complex (miRISC). In Arabidopsis , some MIR genes are expressed in a tissue-specific manner; however, the spatial patterns of MIR gene expression may not be the same as the spatial distribution of miRISCs due to the non-cell autonomous nature of some miRNAs, making it challenging to characterize the spatial profiles of miRNAs. A previous study utilized protoplasting of green fluorescent protein (GFP) marker transgenic lines followed by fluorescence-activated cell sorting (FACS) to isolate cell-type-specific small RNAs. However, the invasiveness of this approach during the protoplasting and cell sorting may stimulate the expression of stress-related miRNAs. To non-invasively profile cell-type-specific miRNAs, we generated transgenic lines in which root cell layer-specific promoters drive the expression of AGO1 and performed immunoprecipitation to non-invasively isolate cell-layer-specific miRISCs. In this protocol, we provide a detailed description of immunoprecipitation of root cell layer-specific GFP-AGO1 using EN7::GFP-AGO1 and ACL5::GFP-AGO1 transgenic plants, followed by small RNA sequencing to profile single-cell-type-specific miRNAs. This protocol is also suitable to profile cell-type-specific miRISCs in other tissues or organs in plants, such as flowers or leaves. This protocol was validated in: Dev Cell (2022), DOI: 10.1016/j.devcel.2022.03.015 Graphical abstract., Competing Interests: Competing interests The authors declare no conflicts of interests., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

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 (β-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. [ABSTRACT FROM AUTHOR]