An AGO10:miR165/6 module regulates meristem activity and xylem development in the Arabidopsis root.

The RNA-silencing effector ARGONAUTE10 influences cell fate in plant shoot and floral meristems. ARGONAUTE10 also accumulates in the root apical meristem (RAM), yet its function(s) therein remain elusive. Here, we show that ARGONAUTE10 is expressed in the root cell initials where it controls overall RAM activity and length. ARGONAUTE10 is also expressed in the stele, where post-transcriptional regulation confines it to the root tip's pro-vascular region. There, variations in ARGONAUTE10 levels modulate metaxylem-vs-protoxylem specification. Both ARGONAUTE10 functions entail its selective, high-affinity binding to mobile miR165/166 transcribed in the neighboring endodermis. ARGONAUTE10-bound miR165/166 is degraded, likely via SMALL-RNA-DEGRADING-NUCLEASES1/2, thus reducing miR165/166 ability to silence, via ARGONAUTE1, the transcripts of cell fate-influencing transcription factors. These include PHABULOSA (PHB), which controls meristem activity in the initials and xylem differentiation in the pro-vasculature. During early germination, PHB transcription increases while dynamic, spatially-restricted transcriptional and post-transcriptional mechanisms reduce and confine ARGONAUTE10 accumulation to the provascular cells surrounding the newly-forming xylem axis. Adequate miR165/166 concentrations are thereby channeled along the ARGONAUTE10-deficient yet ARGONAUTE1-proficient axis. Consequently, inversely-correlated miR165/166 and PHB gradients form preferentially along the axis despite ubiquitous PHB transcription and widespread miR165/166 delivery inside the whole vascular cylinder.

The plant siRNA landscape.

Whereas micro (mi)RNAs are considered the clean, noble side of the small RNA world, small interfering (si)RNAs are often seen as a noisy set of molecules whose barbarian acronyms reflect a large diversity of often elusive origins and functions. Twenty-five years after their discovery in plants, however, new classes of siRNAs are still being identified, sometimes in discrete tissues or at particular developmental stages, making the plant siRNA world substantially more complex and subtle than originally anticipated. Focusing primarily on the model Arabidopsis, we review here the plant siRNA landscape, including transposable elements (TE)-derived siRNAs, a vast array of non-TE-derived endogenous siRNAs, as well as exogenous siRNAs produced in response to invading nucleic acids such as viruses or transgenes. We primarily emphasize the extraordinary sophistication and diversity of their biogenesis and, secondarily, the variety of their known or presumed functions, including via non-cell autonomous activities, in the sporophyte, gametophyte, and shortly after fertilization.

Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway.

Unlike in metazoans, plant microRNAs (miRNAs) undergo stepwise nuclear maturation before engaging cytosolic, sequence-complementary transcripts in association with the silencing effector protein ARGONAUTE1 (AGO1). Since their discovery, how and under which form plant miRNAs translocate to the cytosol has remained unclear, as has their sub-cellular AGO1 loading site(s). Here, we show that the N termini of all plant AGO1s contain a nuclear-localization (NLS) and nuclear-export signal (NES) that, in Arabidopsis thaliana (At), enables AtAGO1 nucleo-cytosolic shuttling in a Leptomycin-B-inhibited manner, diagnostic of CRM1(EXPO1)/NES-dependent nuclear export. Nuclear-only AtAGO1 contains the same 2'O-methylated miRNA cohorts as its nucleo-cytosolic counterpart, but it preferentially interacts with the miRNA loading chaperone HSP90. Furthermore, mature miRNA translocation and miRNA-mediated silencing both require AtAGO1 nucleo-cytosolic shuttling. These findings lead us to propose a substantially revised view of the plant miRNA pathway in which miRNAs are matured, methylated, loaded into AGO1 in the nucleus, and exported to the cytosol as AGO1:miRNA complexes in a CRM1(EXPO1)/NES-dependent manner.

HASTY, the Arabidopsis EXPORTIN5 ortholog, regulates cell-to-cell and vascular microRNA movement.

Plant microRNAs (miRNAs) guide cytosolic post-transcriptional gene silencing of sequence-complementary transcripts within the producing cells, as well as in distant cells and tissues. Here, we used an artificial miRNA-based system (amiRSUL) in Arabidopsis thaliana to explore the still elusive mechanisms of inter-cellular miRNA movement via forward genetics. This screen identified many mutant alleles of HASTY (HST), the ortholog of mammalian EXPORTIN5 (XPO5) with a recently reported role in miRNA biogenesis in Arabidopsis. In both epidermis-peeling and grafting assays, amiRSUL levels were reduced much more substantially in miRNA-recipient tissues than in silencing-emitting tissues. We ascribe this effect to HST controlling cell-to-cell and phloem-mediated movement of the processed amiRSUL, in addition to regulating its biogenesis. While HST is not required for the movement of free GFP or siRNAs, its cell-autonomous expression in amiRSUL-emitting tissues suffices to restore amiRSUL movement independently of its nucleo-cytosolic shuttling activity. By contrast, HST is dispensable for the movement and activity of amiRSUL within recipient tissues. Finally, HST enables movement of endogenous miRNAs that display mostly unaltered steady-state levels in hst mutant tissues. We discuss a role for HST as a hitherto unrecognized regulator of miRNA movement in relation to its recently assigned nuclear function at the nexus of MIRNA transcription and miRNA processing.

LINE-1 activity in facultative heterochromatin formation during X chromosome inactivation.

During X chromosome inactivation (XCI), Xist RNA coats and silences one of the two X chromosomes in female cells. Little is known about how XCI spreads across the chromosome, although LINE-1 elements have been proposed to play a role. Here we show that LINEs participate in creating a silent nuclear compartment into which genes become recruited. A subset of young LINE-1 elements, however, is expressed during XCI, rather than being silenced. We demonstrate that such LINE expression requires the specific heterochromatic state induced by Xist. These LINEs often lie within escape-prone regions of the X chromosome, but close to genes that are subject to XCI, and are associated with putative endo-siRNAs. LINEs may thus facilitate XCI at different levels, with silent LINEs participating in assembly of a heterochromatic nuclear compartment induced by Xist, and active LINEs participating in local propagation of XCI into regions that would otherwise be prone to escape.

A contemporary reassessment of the enhanced transient expression system based on the tombusviral silencing suppressor protein P19.

Transient transgenic expression accelerates pharming and facilitates protein studies in plants. One embodiment of the approach involves leaf infiltration of Agrobacterium strains whose T-DNA is engineered with the gene(s) of interest. However, gene expression during 'agro-infiltration' is intrinsically and universally impeded by the onset of post-transcriptional gene silencing (PTGS). Nearly 20 years ago, a simple method was developed, whereby co-expression of the tombusvirus-encoded P19 protein suppresses PTGS and thus enhances transient gene expression. Yet, how PTGS is activated and suppressed by P19 during the process has remained unclear to date. Here, we address these intertwined questions in a manner also rationalizing how vastly increased protein yields are achieved using a minimal viral replicon as a transient gene expression vector. We also explore, in side-by-side analyses, why some proteins do not accumulate to the expected high levels in the assay, despite vastly increased mRNA levels. We validate that enhanced co-expression of multiple constructs is achieved within the same transformed cells, and illustrate how the P19 system allows rapid protein purification for optimized downstream in vitro applications. Finally, we assess the suitability of the P19 system for subcellular localization studies - an originally unanticipated, yet increasingly popular application - and uncover shortcomings of this specific implement. In revisiting the P19 system using contemporary knowledge, this study sheds light onto its hitherto poorly understood mechanisms while further illustrating its versatility but also some of its limits.

In planta dynamics, transport biases, and endogenous functions of mobile siRNAs in Arabidopsis.

In RNA interference (RNAi), small interfering RNAs (siRNAs) produced from double-stranded RNA guide ARGONAUTE (AGO) proteins to silence sequence-complementary RNA/DNA. RNAi can propagate locally and systemically in plants, but despite recent advances in our understanding of the underlying mechanisms, basic questions remain unaddressed. For instance, RNAi is inferred to diffuse through plasmodesmata (PDs), yet how its dynamics in planta compares with that of established symplastic diffusion markers remains unknown. Also is why select siRNA species, or size classes thereof, are apparently recovered in RNAi recipient tissues, yet only under some experimental settings. Shootward movement of endogenous RNAi in micro-grafted Arabidopsis is also yet to be achieved, while potential endogenous functions of mobile RNAi remain scarcely documented. Here, we show (i) that temporal, localized PD occlusion in source leaves' companion cells (CCs) suffices to abrogate all systemic manifestations of CC-activated mobile transgene silencing, including in sink leaves; (ii) that the presence or absence of specific AGOs in incipient/traversed/recipient tissues likely explains the apparent siRNA length selectivity observed upon vascular movement; (iii) that stress enhancement allows endo-siRNAs of a single inverted repeat (IR) locus to translocate against the shoot-to-root phloem flow; and (iv) that mobile endo-siRNAs generated from this locus have the potential to regulate hundreds of transcripts. Our results close important knowledge gaps, rationalize previously noted inconsistencies between mobile RNAi settings, and provide a framework for mobile endo-siRNA research.

Innate, translation-dependent silencing of an invasive transposon in Arabidopsis.

Co-evolution between hosts' and parasites' genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence degeneration, and, ultimately, loss of autonomy of most transposable elements (TEs). Recognition of newly invasive plant TEs, by contrast, involves an innate antiviral-like silencing response. To investigate this response's activation, we studied the single-copy element EVADÉ (EVD), one of few representatives of the large Ty1/Copia family able to proliferate in Arabidopsis when epigenetically reactivated. In Ty1/Copia elements, a short subgenomic mRNA (shGAG) provides the necessary excess of structural GAG protein over the catalytic components encoded by the full-length genomic flGAG-POL. We show here that the predominant cytosolic distribution of shGAG strongly favors its translation over mostly nuclear flGAG-POL. During this process, an unusually intense ribosomal stalling event coincides with mRNA breakage yielding unconventional 5'OH RNA fragments that evade RNA quality control. The starting point of sRNA production by RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6), exclusively on shGAG, occurs precisely at this breakage point. This hitherto-unrecognized "translation-dependent silencing" (TdS) is independent of codon usage or GC content and is not observed on TE remnants populating the Arabidopsis genome, consistent with their poor association, if any, with polysomes. We propose that TdS forms a primal defense against EVD de novo invasions that underlies its associated sRNA pattern.

Origin, biogenesis, and activity of plant microRNAs.

MicroRNAs (miRNAs) are key posttranscriptional regulators of eukaryotic gene expression. Plants use highly conserved as well as more recently evolved, species-specific miRNAs to control a vast array of biological processes. This Review discusses current advances in our understanding of the origin, biogenesis, and mode of action of plant miRNAs and draws comparisons with their metazoan counterparts.

Genome-scale, single-cell-type resolution of microRNA activities within a whole plant organ.

Loaded into ARGONAUTE(AGO) proteins, eukaryotic micro(mi)RNAs regulate gene expression via cleavage, translational repression, and/or accelerated decay of sequence-complementary target transcripts. Despite their importance in development, cell identity maintenance and stress responses, how individual miRNAs contribute to spatial gene regulation within the complex cell mosaics formed in tissues/organs has remained inaccessible in any organism to date. We have developed a non-invasive methodology to examine, at single-cell-type resolution, the AGO-loading and activity patterns of entire miRNA cohorts in intact organs, applied here to the Arabidopsis root tip. A dual miRNAome-targetome analytical interface allowing intuitive data integration/visualization was developed as the basis for in-depth investigations via single-cell-type experimentation. These uncovered an array of so far speculative or hitherto unknown types of spatial miRNA-mediated gene regulation schemes, including via widespread cell-to-cell movement between contiguous layers of distinct identities. This study provides the proof of principle that minimally invasive, genome-scale analysis of miRNA activities within and between single-cell types of whole organs is achievable.

A universal method for the rapid isolation of all known classes of functional silencing small RNAs.

Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method - coined 'TraPR' for Trans-kingdom, rapid, affordable Purification of RISCs - operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequencing libraries outperforming those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.

Functional characterization of Arabidopsis ARGONAUTE 3 in reproductive tissues.

Arabidopsis encodes 10 ARGONAUTE (AGO) effectors of RNA silencing, canonically loaded with either 21-22 nucleotide (nt) long small RNAs (sRNAs) to mediate post-transcriptional gene silencing (PTGS) or 24 nt sRNAs to promote RNA-directed DNA methylation. Using full-locus constructs, we characterized the expression, biochemical properties and possible modes of action of AGO3. Although AGO3 arose from a recent duplication at the AGO2 locus, their expression patterns differ drastically, with AGO2 being expressed in both male and female gametes whereas AGO3 accumulates in aerial vascular terminations and specifically in chalazal seed integuments. Accordingly, AGO3 downregulation alters gene expression in siliques. Similar to AGO2, AGO3 binds sRNAs with a strong 5' adenosine bias, but unlike Arabidopsis AGO2, it binds 24 nt sRNAs most efficiently. AGO3 immunoprecipitation experiments in siliques revealed that these sRNAs mostly correspond to genes and intergenic regions in a manner reflecting their respective accumulation from their loci of origin. AGO3 localizes to the cytoplasm and co-fractionates with polysomes to possibly mediate PTGS via translation inhibition.

Extensive profiling in Arabidopsis reveals abundant polysome-associated 24-nt small RNAs including AGO5-dependent pseudogene-derived siRNAs.

In a reductionist perspective, plant silencing small (s)RNAs are often classified as mediating nuclear transcriptional gene silencing (TGS) or cytosolic posttranscriptional gene silencing (PTGS). Among the PTGS diagnostics is the association of AGOs and their sRNA cargos with the translation apparatus. In Arabidopsis, this is observed for AGO1 loaded with micro(mi)RNAs and, accordingly, translational-repression (TR) is one layer of plant miRNA action. Using AGO1:miRNA-mediated TR as a paradigm, we explored, with two unrelated polysome-isolation methods, which, among the ten Arabidopsis AGOs and numerous sRNA classes, interact with translation. We found that representatives of all three AGO-clades associate with polysomes, including the TGS-effector AGO4 and stereotypical 24-nt sRNAs that normally mediate TGS of transposons/repeats. Strikingly, approximately half of these annotated 24-nt siRNAs displayed unique matches in coding regions/introns of genes, and in pseudogenes, but not in transposons/repeats commonly found in their vicinity. Protein-coding gene-derived 24-nt sRNAs correlate with gene-body methylation. Those derived from pseudogenes belong to two main clusters defined by their parental-gene expression patterns, and are vastly enriched in AGO5, itself found on polysomes. Based on their tight expression pattern in developing and mature siliques, their biogenesis, and genomic/epigenomic features of their loci-of-origin, we discuss potential roles for these hitherto unknown polysome-enriched, pseudogene-derived siRNAs.

Chemical enhancers of posttranscriptional gene silencing in Arabidopsis.

RNAi mediated by small-interfering RNAs (siRNAs) operates via transcriptional (TGS) and posttranscriptional gene silencing (PTGS). In Arabidopsis thaliana, TGS relies on DICER-LIKE-3 (DCL3)-dependent 24-nt siRNAs loaded into AGO4-clade ARGONAUTE effector proteins. PTGS operates via DCL4-dependent 21-nt siRNAs loaded into AGO1-clade proteins. We set up and validated a medium-throughput, semi-automatized procedure enabling chemical screening, in a 96-well in vitro format, of Arabidopsis transgenic seedlings expressing an inverted-repeat construct from the phloem companion cells. The ensuing quantitative PTGS phenotype was exploited to identify molecules, which, upon topical application, either inhibit or enhance siRNA biogenesis/activities. The vast majority of identified modifiers were enhancers, among which Sortin1, Isoxazolone, and [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) provided the most robust and consistent results, including upon their application onto soil-grown plants in which their effect was nonautonomous and long lasting. The three molecules increased the RNAi potency of the inverted-repeat construct, in large part by enhancing 21-nt siRNA accumulation and loading into AGO1, and concomitantly reducing AGO4 and DCL3 levels in planta. A similar, albeit not identical effect, was observed on 22-nt siRNAs produced from a naturally occurring inverted-repeat locus, demonstrating that the molecules also enhance endogenous PTGS. In standardized assays conducted in seedling extracts, the three enhancers selectively increased DCL4-mediated processing of in vitro-synthesized double-stranded RNAs, indicating the targeting of a hitherto unknown PTGS component probably independent of the DCL4-cofactor DOUBLE-STRANDED RNA-BINDING 4 (DRB4). This study establishes the proof-of-concept that RNAi efficacy can be modulated by chemicals in a whole organism. Their potential applications and the associated future research are discussed.

A Suppressor Screen for AGO1 Degradation by the Viral F-Box P0 Protein Uncovers a Role for AGO DUF1785 in sRNA Duplex Unwinding.

In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing and is a key component in antiviral responses. The polerovirus F-box P0 protein triggers AGO1 degradation as a viral counterdefense. Here, we identified a motif in AGO1 that is required for its interaction with the S phase kinase-associated protein1-cullin 1-F-box protein (SCF) P0 (SCFP0) complex and subsequent degradation. The AGO1 P0 degron is conserved and confers P0-mediated degradation to other AGO proteins. Interestingly, the degron motif is localized in the DUF1785 domain of AGO1, in which a single point mutation (ago1-57, obtained by forward genetic screening) compromises recognition by SCFP0 Recapitulating formation of the RNA-induced silencing complex in a cell-free system revealed that this mutation impairs RNA unwinding, leading to stalled forms of AGO1 still bound to double-stranded RNAs. In vivo, the DUF1785 is required for unwinding perfectly matched siRNA duplexes, but is mostly dispensable for unwinding imperfectly matched miRNA duplexes. Consequently, its mutation nearly abolishes phased siRNA production and sense transgene posttranscriptional gene silencing. Overall, our work sheds new light on the mode of AGO1 recognition by P0 and the in vivo function of DUF1785 in RNA silencing.

Revisiting the principles of microRNA target recognition and mode of action.

MicroRNAs (miRNAs) are fundamental regulatory elements of animal and plant gene expression. Although rapid progress in our understanding of miRNA biogenesis has been achieved by experimentation, computational approaches have also been influential in determining the general principles that are thought to govern miRNA target recognition and mode of action. We discuss how these principles are being progressively challenged by genetic and biochemical studies. In addition, we discuss the role of target-site-specific endonucleolytic cleavage, which is the hallmark of experimental RNA interference and a mechanism that is used by plant miRNAs and a few animal miRNAs. Generally thought to be merely a degradation mechanism, we propose that this might also be a biogenesis mechanism for biologically functional, non-coding RNA fragments.