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RNA helicases (RHs) are a family of ubiquitous enzymes that alter RNA structures and remodel ribonucleoprotein complexes typically using energy from the hydrolysis of ATP. RHs are involved in various aspects of RNA processing and metabolism, exemplified by transcriptional regulation, pre-mRNA splicing, miRNA biogenesis, liquid–liquid phase separation, and rRNA biogenesis, among other molecular processes. Through these mechanisms, RHs contribute to vegetative and reproductive growth, as well as abiotic and biotic stress responses throughout the life cycle in plants. In this review, we systematically characterize RH-featured domains and signature motifs in Arabidopsis. We also summarize the functions and mechanisms of RHs in various biological processes in plants with a focus on DEAD-box and DEAH-box RNA helicases, aiming to present the latest understanding of RHs in plant biology. [ABSTRACT FROM AUTHOR]

The majority of the genome is transcribed to RNA in living organisms. RNA transcripts can form astonishing arrays of secondary and tertiary structures via Watson–Crick, Hoogsteen, or wobble base pairing. In vivo , RNA folding is not a simple thermodynamic event of minimizing free energy. Instead, the process is constrained by transcription, RNA-binding proteins, steric factors, and the microenvironment. RNA secondary structure (RSS) plays myriad roles in numerous biological processes, such as RNA processing, stability, transportation, and translation in prokaryotes and eukaryotes. Emerging evidence has also implicated RSS in RNA trafficking, liquid–liquid phase separation, and plant responses to environmental variations such as temperature and salinity. At molecular level, RSS is correlated with splicing, polyadenylation, protein synthesis, and miRNA biogenesis and functions. In this review, we summarize newly reported methods for probing RSS in vivo and functions and mechanisms of RSS in plant physiology. [ABSTRACT FROM AUTHOR]

Plant viruses have evolved multiple strategies to overcome host defense to establish an infection. Here, we identified two components of a host mitogen-activated protein kinase (MAPK) cascade, MKK2 and MPK4, as bona fide targets of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNV). βC1 interacts with the kinase domain of MKK2 and inhibits its activity. In vivo, βC1 suppresses flagellin-induced MAPK activation and downstream responses by targeting MKK2. Furthermore, βC1 also interacts with MPK4 and inhibits its kinase activity. TYLCCNV infection induces the activation of the MAPK cascade, mutation in MKK2 or MPK4 renders the plant more susceptible to TYLCCNV, and can complement the lack of βC1. This work shows for the first time that a plant virus both activates and suppress a MAPK cascade, and the discovery of the ability of βC1 to selectively interfere with the host MAPK activation illustrates a novel virulence function and counter-host defense mechanism of geminiviruses. [ABSTRACT FROM AUTHOR]

Highlights • miRNAs are likely loaded into AGO1 in the nucleus. • RICEs degrade the uridylated AGO-cleaved 5′ mRNA fragments to maintain RISC. • Nuclear AGO1 can regulate transcription and DNA repair. • AGO3 functions like the clade of AGO4/6/9 in the RdDM pathway. • AGO10 not only sequesters miRNAs but also promotes their degradation by SDN1. Argonaute (AGO) proteins are the key effector of RNA-induced silencing complex (RISC). Land plants typically encode numerous AGO proteins, and they can be typically divided into two major functional groups based on the species of their housed small RNAs (sRNAs). One group of AGOs, guided by 24-nucleotide (nt) sRNAs, canonically function in nuclei to implement transcriptional gene silencing (TGS), whereas the other group of AGOs, guided by 21-nt sRNAs, act in the cytoplasm to fulfill posttranscriptional gene silencing (PTGS). Many new discoveries have been recently made on functions and mechanisms of AGO proteins in plants, and some of the findings change our views on the conventional classification and roles of AGO proteins. In this review, we summarize our current knowledge of AGO proteins in plants. [ABSTRACT FROM AUTHOR]

The whitefly-transmitted geminiviruses induce severe developmental abnormalities in plants. Geminivirus-encoded C4 protein functions as one of viral symptom determinants that could induce abnormal cell division. However, the molecular mechanism by which C4 contributes to cell division induction remains unclear. Here we report that tomato leaf curl Yunnan virus (TLCYnV) C4 interacts with a glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase, designed NbSKη, in Nicotiana benthamiana. Pro32, Asn34 and Thr35 of TLCYnV C4 are critical for its interaction with NbSKη and required for C4-induced typical symptoms. Interestingly, TLCYnV C4 directs NbSKη to the membrane and reduces the nuclear-accumulation of NbSKη. The relocalization of NbSKη impairs phosphorylation dependent degradation on its substrate-Cyclin D1.1 (NbCycD1;1), thereby increasing the accumulation level of NbCycD1;1 and inducing the cell division. Moreover, NbSKη-RNAi, 35S::NbCycD1;1 transgenic N. benthamiana plants have the similar phenotype as 35S::C4 transgenic N. benthamiana plants on callus-like tissue formation resulted from abnormal cell division induction. Thus, this study provides new insights into mechanism of how a viral protein hijacks NbSKη to induce abnormal cell division in plants. [ABSTRACT FROM AUTHOR]

Like metazoans, plants use small regulatory RNAs ( sRNAs) to direct gene expression. Several classes of sRNAs, which are distinguished by their origin and biogenesis, exist in plants. Among them, micro RNAs (mi RNAs) and trans-acting small interfering RNAs (ta-si RNAs) mainly inhibit gene expression at post-transcriptional levels. In the past decades, plant mi RNAs and ta-si RNAs have been shown to be essential for numerous developmental processes, including growth and development of shoots, leaves, flowers, roots and seeds, among others. In addition, mi RNAs and ta-si RNAs are also involved in the plant responses to abiotic and biotic stresses, such as drought, temperature, salinity, nutrient deprivation, bacteria, virus and others. This review summarizes the roles of mi RNAs and ta-si RNAs in plant physiology and development. [ABSTRACT FROM AUTHOR]

Global climate change, increasingly erratic weather and a burgeoning global population are significant threats to the sustainability of future crop production. There is an urgent need for the development of robust measures that enable crops to withstand the uncertainty of climate change whilst still producing maximum yields. Resurrection plants possess the unique ability to withstand desiccation for prolonged periods, can be restored upon watering and represent great potential for the development of stress tolerant crops. Here, we describe the remarkable stress characteristics of Tripogon loliiformis, an uncharacterised resurrection grass and close relative of the economically important cereals, rice, sorghum, and maize. We show that T. loliiformis survives extreme environmental stress by implementing autophagy to prevent Programmed Cell Death. Notably, we identified a novel role for trehalose in the regulation of autophagy in T.loliiformis. Transcriptome, Gas Chromatography Mass Spectrometry, immunoblotting and confocal microscopy analyses directly linked the accumulation of trehalose with the onset of autophagy in dehydrating and desiccated T. loliiformis shoots. These results were supported in vitro with the observation of autophagosomes in trehalose treated T. loliiformis leaves; autophagosomes were not detected in untreated samples. Presumably, once induced, autophagy promotes desiccation tolerance in T.loliiformis, by removal of cellular toxins to suppress programmed cell death and the recycling of nutrients to delay the onset of senescence. These findings illustrate how resurrection plants manipulate sugar metabolism to promote desiccation tolerance and may provide candidate genes that are potentially useful for the development of stress tolerant crops. [ABSTRACT FROM AUTHOR]

Summary Arabidopsis Argonaute10 (AGO10) specifically sequesters miR165 and miR166 and antagonizes their activity, thus regulating shoot apical meristem (SAM) development. However, where and when this sequestration acts is currently unclear. We show here that AGO10 represses miR165/166 activity in the embryo proper during early embryogenesis, through the apical and central regions of mature embryos, and eventually in the entire adaxial domain and vasculature of the cotyledons and leaf primordia. These locations are essentially identical to regions expressing PHABULOSA and REVOLUTA , mRNA targets of miR165/166. The Arabidopsis genome contains nine MIR165/166 genes. Sequestration of miR165/166 by the MIR165b , MIR166a , MIR166b , and MIR166g promoters efficiently rescues the SAM defect in ago10 mutants. Comparison of the expression patterns of AGO10 and the four MIR165/166 members suggests that AGO10 quenches the non-cell-autonomous activity of any miR165/166 that moves into AGO10 -expressing niches. Thus, this study provides insight into how the spatiotemporal regulation of AGO10-miR165/166 activity affects SAM development. [ABSTRACT FROM AUTHOR]

Trajectory inference (TI) or pseudotime analysis has dramatically extended the analytical framework of single-cell RNA-seq data, allowing regulatory genes contributing to cell differentiation and those involved in various dynamic cellular processes to be identified. However, most TI analysis procedures deal with individual genes independently while overlooking the regulatory relations between genes. Integrating information from gene regulatory networks (GRNs) at different pseudotime points may lead to more interpretable TI results. To this end, we introduce scInTime—an unsupervised machine learning framework coupling inferred trajectory with single-cell GRNs (scGRNs) to identify master regulatory genes. We validated the performance of our method by analyzing multiple scRNA-seq data sets. In each of the cases, top-ranking genes predicted by scInTime supported their functional relevance with corresponding signaling pathways, in line with the results of available functional studies. Overall results demonstrated that scInTime is a powerful tool to exploit pseudotime-series scGRNs, allowing for a clear interpretation of TI results toward more significant biological insights. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) originate from primary transcripts (pri-miRNAs) with characteristic stem-loop structures, and their accurate processing is required for the production of functional miRNAs. Here, using the pri-miR-166 family in Arabidopsis thaliana as a paradigm, we report the crucial role of pri-miRNA terminal loops in miRNA biogenesis. We found that multibranched terminal loops in pri-miR-166s substantially suppress miR-166 expression in vivo. Unlike canonical processing of pri-miRNAs, terminal loop-branched pri-miRNAs can be processed by Dicer-like 1 (DCL1) complexes bidirectionally from base to loop and from loop to base, resulting in productive and abortive processing of miRNAs, respectively. In both cases, DCL1 complexes canonically cut pri-miRNAs at a distance of 16-17 bp from a reference single-stranded loop region. DCL1 also adjusts processing sites toward an internal loop through its helicase domain. These results provide new insight into the poorly understood processing mechanism of pri-miRNAs with complex secondary structures. [ABSTRACT FROM AUTHOR]

Plant stem cells in the shoot apical meristem (SAM) possess the unique abilities of both self-renewal for SAM maintenance and providing undifferentiated daughter cells for initiation and subsequent development of aerial organs. The coordination between stem cell renewal and cell differentiation during organogenesis is regulated by elaborate genetic pathways involving numerous transcription factors and other molecules. In the past decade, microRNAs (miRNAs) have emerged as pivotal regulators in many biological processes including meristem homeostasis and differentiation in plants. In this review, we summarize current knowledge about the function and mechanism of a family of miRNAs (miR165/166), the miRNA-designated Argonautes (AGOs), and the miRNA-regulated targets in SAM development and maintenance. [ABSTRACT FROM AUTHOR]

Norwalk virus (NV) is an important agent of epidemic gastroenteritis, and an oral subunit vaccine shows potential for protection. Recombinant Norwalk virus (rNV) capsid protein expressed in plants assembles virus-like particles (VLPs) that are orally immunogenic in mice and humans. In this article we examine rNV expression in tomato and potato using a plant-optimized gene, and test the immunogenicity of dried tomato fruit and potato tuber fed to mice. The synthetic gene increased rNV expression fourfold in tomato and potato plants, which assembled VLP. Four doses of 0.4 g freeze-dried tomato fruit containing 64 µg rNV (40 µg VLPs) induced NV-specific serum IgG and mucosal IgA in ≥ 80% of mice, while doses of 0.8 g elicited systemic and mucosal antibody responses in all mice. Feedings of 1 g freeze-dried potato tuber containing 120 µg rNV (90 µg VLPs) were required to produce 100% responsiveness. Oxidation of phenolic compounds upon rehydration of dried tuber caused significant VLP instability, thus decreasing immunogenicity. Air-dried tomato fruit stimulated stronger immune responses than freeze-dried fruit of the same mass, perhaps by limiting the destruction of plant cell matrix and membrane systems that occurs with freeze-drying. Thus, rNV in dried transgenic tomato fruit was a more potent immunogen than that in dried potato tubers, based on the total VLPs ingested. These findings support the use of stabilized, dried tomato fruit for oral delivery of subunit vaccines. [ABSTRACT FROM AUTHOR]

Drought causes approximately two-thirds of crop and yield loss worldwide. To sustain future generations, there is a need to develop robust crops with enhanced water use efficiency. Resurrection plants are naturally resilient and tolerate up to 95% water loss with the ability to revive upon watering. Stress is genetically encoded and resilient species may garner tolerance by tightly regulating the expression of stress-related genes. MicroRNAs (miRNAs) post-transcriptionally regulate development and other stress response processes in eukaryotes. However, their role in resurrection plant desiccation tolerance is poorly understood. In this study, small RNA sequencing and miRNA expression profiling was conducted using Tripogon loliiformis plants subjected to extreme water deficit conditions. Differentially expressed miRNA profiles, target mRNAs, and their regulatory processes were elucidated. Gene ontology enrichment analysis revealed that development, stress response, and regulation of programmed cell death biological processes; Oxidoreductase and hydrolyase molecular activities; and SPL, MYB, and WRKY transcription factors were targeted by miRNAs during dehydration stress, indicating the indispensable regulatory role of miRNAs in desiccation tolerance. This study provides insights into the molecular mechanisms of desiccation tolerance in the resurrection plant T. loliiformis. This information will be useful in devising strategies for crop improvement on enhanced drought tolerance and water use efficiency. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • DMS-MaPseq was modified for probing genome-wide RNA structure in Arabidopsis. • Procedures for DMS treatment and library preparation were optimized. • Modified DMS-MaPseq produced high-quality and reproducible RNA structure data. • Sequencing depth and coverage for genome-wide DMS-MaPseq were assessed. • Two examples of mRNA structures are shown. Abstract Transcripts have intrinsic propensity to form stable secondary structure that is fundamental to regulate RNA transcription, splicing, translation, RNA localization and turnover. Numerous methods that integrate chemical reactions with next-generation sequencing (NGS) have been applied to study in vivo RNA structure, providing new insights into RNA biology. Dimethyl sulfate (DMS) probing coupled with mutational profiling through NGS (DMS-MaPseq) is a newly developed method for revealing genome-wide or target-specific RNA structure. Herein, we present our experimental protocol of a modified DMS-MaPseq method for plant materials. The DMS treatment condition was optimized, and library preparation procedures were simplified. We also provided custom scripts for bioinformatic analysis of genome-wide DMS-MaPseq data. Bioinformatic results showed that our method could generate high-quality and reproducible data. Further, we assessed sequencing depth and coverage for genome-wide RNA structure profiling in Arabidopsis, and provided two examples of in vivo structure of mobile RNAs. We hope that our modified DMS-MaPseq method will serve as a powerful tool for analyzing in vivo RNA structurome in plants. [ABSTRACT FROM AUTHOR]

Summary: The shoot apical meristem (SAM) comprises a group of undifferentiated cells that divide to maintain the plant meristem and also give rise to all shoot organs. SAM fate is specified by class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, which are targets of miR166/165. In Arabidopsis, AGO10 is a critical regulator of SAM maintenance, and here we demonstrate that AGO10 specifically interacts with miR166/165. The association is determined by a distinct structure of the miR166/165 duplex. Deficient loading of miR166 into AGO10 results in a defective SAM. Notably, the miRNA-binding ability of AGO10, but not its catalytic activity, is required for SAM development, and AGO10 has a higher binding affinity for miR166 than does AGO1, a principal contributor to miRNA-mediated silencing. We propose that AGO10 functions as a decoy for miR166/165 to maintain the SAM, preventing their incorporation into AGO1 complexes and the subsequent repression of HD-ZIP III gene expression. PaperFlick: Display Omitted [Copyright &y& Elsevier]

Summary Serrate (SE) is a key component in RNA metabolism. Little is known about whether and how it can regulate epigenetic silencing. Here, we report histone methyltransferases ATXR5 and ATXR6 (ATXR5/6) as novel partners of SE. ATXR5/6 deposit histone 3 lysine 27 monomethylation (H3K27me1) to promote heterochromatin formation, repress transposable elements (TEs), and control genome stability in Arabidopsis . SE binds to ATXR5/6-regulated TE loci and promotes H3K27me1 accumulation in these regions. Furthermore, SE directly enhances ATXR5 enzymatic activity in vitro . Unexpectedly, se mutation suppresses the TE reactivation and DNA re-replication phenotypes in the atxr5 atxr6 mutant. The suppression of TE expression results from triggering RNA-dependent RNA polymerase 6 (RDR6)-dependent RNA silencing in the se atxr5 atxr6 mutant. We propose that SE facilitates ATXR5/6-mediated deposition of the H3K27me1 mark while inhibiting RDR6-mediated RNA silencing to protect TE transcripts. Hence, SE coordinates epigenetic silencing and RNA processing machineries to fine-tune the TE expression. [ABSTRACT FROM AUTHOR]

RNA secondary structure (RSS) of primary microRNAs (pri-miRNAs) is a key determinant for miRNA production. Here we report that RNA helicase (RH) Brr2a, best known as a spliceosome component, modulates the structural complexity of pri-miRNAs to fine tune miRNA yield. Brr2a interacts with microprocessor component HYL1 and its loss reduces the levels of miRNAs derived from both intron-containing and intron-lacking pri-miRNAs. Brr2a binds to pri-miRNAs in vivo and in vitro. Furthermore, Brr2a hydrolyses ATP and the activity can be significantly enhanced by pri-miRNAs. Consequently, Brr2a unwinds pri-miRNAs in vitro. Moreover, Brr2a variants with compromised ATPase or RH activity are incapable of unwinding pri-miRNA, and their transgenic plants fail to restore miRNA levels in brr2a-2. Importantly, most of tested pri-miRNAs display distinct RSS, rendering them unsuitable for efficient processing in brr2a mutants vs Col-0. Collectively, this study reveals that Brr2a plays a non-canonical role in miRNA production beyond splicing regulation., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Methyltransferase complex (MTC) deposits N 6-adenosine (m 6 A) onto RNA, whereas microprocessor produces miRNA. Whether and how these two distinct complexes cross-regulate each other has been poorly studied. Here we report that the MTC subunit B (MTB) tends to form insoluble condensates with poor activity, with its level monitored by 20S proteasome. Conversely, the microprocessor component SERRATE (SE) forms liquid-like condensates, which in turn promotes solubility and stability of MTB, leading to increased MTC activity. Consistently, the hypomorphic lines expressing SE variants, defective in MTC interaction or liquid-like phase behavior, exhibit reduced m 6 A level. Reciprocally, MTC can recruit microprocessor to MIRNA loci, prompting co-transcriptional cleavage of primary miRNA (pri-miRNAs) substrates. Additionally, pri-miRNAs carrying m 6 A modifications at their single-stranded basal regions are enriched by m 6 A readers, which retain microprocessor in the nucleoplasm for continuing processing. This reveals an unappreciated mechanism of phase separation in RNA modification and processing through MTC and microprocessor coordination.

Intrinsically disordered proteins (IDPs) SAID1/2 are hypothetic dentin sialophosphoprotein-like proteins, but their true functions are unknown. Here, we identified SAID1/2 as negative regulators of SERRATE (SE), a core factor in miRNA biogenesis complex (microprocessor). Loss-of-function double mutants of said1; said2 caused pleiotropic developmental defects and thousands of differentially expressed genes that partially overlapped with those in se. said1; said2 also displayed increased assembly of microprocessor and elevated accumulation of microRNAs (miRNAs). Mechanistically, SAID1/2 promote pre-mRNA processing 4 kinase A-mediated phosphorylation of SE, causing its degradation in vivo. Unexpectedly, SAID1/2 have strong binding affinity to hairpin-structured pri-miRNAs and can sequester them from SE. Moreover, SAID1/2 directly inhibit pri-miRNA processing by microprocessor in vitro. Whereas SAID1/2 did not impact SE subcellular compartmentation, the proteins themselves exhibited liquid-liquid phase condensation that is nucleated on SE. Thus, we propose that SAID1/2 reduce miRNA production through hijacking pri-miRNAs to prevent microprocessor activity while promoting SE phosphorylation and its destabilization in Arabidopsis.

Phosphorylation can quickly switch on/off protein functions. Here, we reported pre-mRNA processing 4 kinase A (PRP4KA), and its paralogs interact with Serrate (SE), a key factor in RNA processing. PRP4KA phosphorylates at least five residues of SE in vitro and in vivo. Hypophosphorylated, but not hyperphosphorylated, SE variants could readily rescue se phenotypes in vivo. Moreover, hypophosphorylated SE variants had stronger binding affinity to microprocessor component HYL1 and were more resistant to degradation by 20 S proteasome than hyperphosphorylated counterparts. Knockdown of the kinases enhanced the accumulation of hypophosphorylated SE. However, the excessive SE interfered with the assembly and function of SE-scaffolded macromolecule complexes, causing the se -like defects in the mutant and wild-type backgrounds. Thus, phosphorylation of SE via PRP4KA can quickly clear accumulated SE to secure its proper amount. This study provides new insight into how protein phosphorylation regulates miRNA metabolism through controlling homeostasis of SE accumulation in plants.

Posttranscriptional gene silencing (PTGS) is a regulatory mechanism to suppress undesired transcripts. Here, we identified Flowering locus VE ( FVE ), a well-known epigenetic component, as a new player in cytoplasmic PTGS. Loss-of-function fve mutations substantially reduced the accumulation of transgene-derived small interfering RNAs (siRNAs). FVE interacts with suppressor of gene silencing 3 (SGS3), a master component in PTGS. FVE promotes SGS3 homodimerization that is essential for its function. FVE can bind to single-stranded RNA and double-stranded RNA (dsRNA) with moderate affinities, while its truncated form FVE-8 has a significantly increased binding affinity to dsRNA. These affinities affect the association and channeling of SGS3-RNA to downstream dsRNA binding protein 4 (DRB4)/Dicer-like protein 2/4 (DCL2/4) complexes. Hence, FVE, but not FVE-8, biochemically enhances the DRB4/DCL2/4 activity in vitro. We surmise that FVE promotes production of transgene-derived siRNAs through concertedly tuning SGS3-DRB4/DCL2/4 functions. Thus, this study revealed a noncanonical role of FVE in PTGS., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be controlled by the 20S proteasome. Relatively little is known about the specific functions of the 20S proteasome and the regulatory mechanisms of IDP degradation in plants compared to other species because there is a lack of systematic protocols for in vitro assembly of this complex to perform in vitro degradation assays. Here, we present a detailed protocol of in vitro reconstitution assay of the 20S proteasome in Arabidopsis by modifying previously reported methods. The main strategy to obtain the 20S core proteasome here is to strip away the 19S regulatory subunits from the 26S proteasome. The protocol has two major parts: 1) Affinity purification of 20S proteasomes from stable transgenic lines expressing epitope-tagged PAG1, an essential component of the 20S proteasome (Procedures A-D) and 2) an in vitro 20S proteasome degradation assay (Procedure E). We anticipate that these protocols will provide simple and effective approaches to study in vitro degradation by the 20S proteasome and advance the study of protein metabolism in plants., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

RNA silencing plays a critical role in diverse biological processes in plants including growth, development, and responses to abiotic and biotic stresses. RNA silencing is guided by small non-coding RNAs (sRNAs) with the length of 21-24 nucleotides (nt) that are loaded into Argonaute (AGO) to repress expression of target loci and transcripts through transcriptional or posttranscriptional gene silencing mechanisms. Identification and quantitative characterization of sRNAs are crucial steps toward appreciation of their functions in biology. Here, we developed a step-by-step protocol to precisely illustrate the process of cloning of sRNA libraries and correspondingly computational analysis of the recovered sRNAs. This protocol can be used in all kinds of organisms, including Arabidopsis, and is compatible with various high-throughput sequence technologies such as Illumina Hiseq. Thus, we wish that this protocol represents an accurate way to identify and quantify sRNAs in vivo.

Trypanosome U-insertion/deletion RNA editing in mitochondrial mRNAs involves guide RNAs (gRNAs) and the auxiliary RNA editing substrate binding complex (RESC) and RNA editing helicase 2 complex (REH2C). RESC and REH2C stably copurify with editing mRNAs but the functional interplay between these complexes remains unclear. Most steady-state mRNAs are partially edited and include misedited "junction" regions that match neither pre-mRNA nor fully edited transcripts. Editing specificity is central to mitochondrial RNA maturation and function, but its basic control mechanisms remain unclear. Here we applied a novel nucleotide-resolution RNA-seq approach to examine ribosomal protein subunit 12 (RPS12) and ATPase subunit 6 (A6) mRNA transcripts. We directly compared transcripts associated with RESC and REH2C to those found in total mitochondrial RNA. RESC-associated transcripts exhibited site-preferential enrichments in total and accurate edits. REH2C loss-of-function induced similar substrate-specific and site-specific editing effects in total and RESC-associated RNA. It decreased total editing primarily at RPS12 5' positions but increased total editing at examined A6 3' positions. REH2C loss-of-function caused site-preferential loss of accurate editing in both transcripts. However, changes in total or accurate edits did not necessarily involve common sites. A few 5' nucleotides of the initiating gRNA (gRNA-1) directed accurate editing in both transcripts. However, in RPS12, two conserved 3'-terminal adenines in gRNA-1 could direct a noncanonical 2U-insertion that causes major pausing in 3'-5' progression. In A6, a noncanonical sequence element that depends on REH2C in a region normally targeted by the 3' half of gRNA-1 may hinder early editing progression. Overall, we defined transcript-specific effects of REH2C loss., (© 2020 Kumar et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The advent of host-induced gene silencing (HIGS) technology for the development of pathogen-resistant cultivars led to the discovery of cross-kingdom RNA interference. In a recent Science paper, Cai et al. (2018) discovered that plant extracellular vesicles act as Trojan horses to deliver small RNAs into fungi to fight infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Argonaute (AGO) proteins execute RNA-induced transcriptional and post-transcriptional gene silencing. In this issue of Developmental Cell, Liu et al. (2018) uncover a nuclear function for Arabidopsis AGO1 in positively regulating gene expression. AGO1, guided by small RNAs, binds to chromatin to induce target gene transcription in response to environmental stimuli., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Transition from vegetative to floral buds is a critical physiological change during flower induction that determines fruit productivity. Small non-coding RNAs (sRNAs) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) are pivotal regulators of plant growth and development. Although the key role of sRNAs in flowering regulation has been well-described in Arabidopsis and some other annual plants, their relevance to vegetative-to-floral transition (hereafter, referred to floral transition) in perennial woody trees remains under defined. Here, we performed Illumina sequencing of sRNA libraries prepared from vegetative and floral bud during flower induction of the apple trees. A large number of sRNAs exemplified by 33 previously annotated miRNAs and six novel members display significant differential expression (DE) patterns. Notably, most of these DE-miRNAs in floral transition displayed opposite expression changes in reported phase transition in apple trees. Bioinformatics analysis suggests most of the DE-miRNAs targeted transcripts involved in SQUAMOSA PROMOTER BINDING PROTEIN-LIKE ( SPL ) gene regulation, stress responses, and auxin and gibberellin (GA) pathways, with further suggestion that there is an inherent link between physiological stress response and metabolism reprogramming during floral transition. We also observed significant changes in 24 nucleotide (nt) sRNAs that are hallmarks for RNA-dependent DNA methylation (RdDM) pathway, suggestive of the correlation between epigenetic modifications and the floral transition. The study not only provides new insight into our understanding of fundamental mechanism of poorly studied floral transition in apple and other woody plants, but also presents important sRNA resource for future in-depth research in the apple flowering physiology.

RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5' products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC.

Transcriptional gene silencing (TGS) can serve as an innate immunity against invading DNA viruses throughout Eukaryotes. Geminivirus code for TrAP protein to suppress the TGS pathway. Here, we identified an Arabidopsis H3K9me2 histone methyltransferase, Su(var)3-9 homolog 4/Kryptonite (SUVH4/KYP), as a bona fide cellular target of TrAP. TrAP interacts with the catalytic domain of KYP and inhibits its activity in vitro. TrAP elicits developmental anomalies phenocopying several TGS mutants, reduces the repressive H3K9me2 mark and CHH DNA methylation, and reactivates numerous endogenous KYP-repressed loci in vivo. Moreover, KYP binds to the viral chromatin and controls its methylation to combat virus infection. Notably, kyp mutants support systemic infection of TrAP-deficient Geminivirus. We conclude that TrAP attenuates the TGS of the viral chromatin by inhibiting KYP activity to evade host surveillance. These findings provide new insight on the molecular arms race between host antiviral defense and virus counter defense at an epigenetic level.