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.

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.

Movement and differential consumption of short interfering RNA duplexes underlie mobile RNA interference.

In RNA interference (RNAi), the RNase III Dicer processes long double-stranded RNA (dsRNA) into short interfering RNA (siRNA), which, when loaded into ARGONAUTE (AGO) family proteins, execute gene silencing1. Remarkably, RNAi can act non-cell autonomously2,3: it is graft transmissible4-7, and plasmodesmata-associated proteins modulate its cell-to-cell spread8,9. Nonetheless, the molecular mechanisms involved remain ill defined, probably reflecting a disparity of experimental settings. Among other caveats, these almost invariably cause artificially enhanced movement via transitivity, whereby primary RNAi-target transcripts are converted into further dsRNA sources of secondary siRNA5,10,11. Whether siRNA mobility naturally requires transitivity and whether it entails the same or distinct signals for cell-to-cell versus long-distance movement remains unclear, as does the identity of the mobile signalling molecules themselves. Movement of long single-stranded RNA, dsRNA, free/AGO-bound secondary siRNA or primary siRNA have all been advocated12-15; however, an entity necessary and sufficient for all known manifestations of plant mobile RNAi remains to be ascertained. Here, we show that the same primary RNAi signal endows both vasculature-to-epidermis and long-distance silencing movement from three distinct RNAi sources. The mobile entities are AGO-free primary siRNA duplexes spreading length and sequence independently. However, their movement is accompanied by selective siRNA depletion reflecting the AGO repertoires of traversed cell types. Coupling movement with this AGO-mediated consumption process creates qualitatively distinct silencing territories, potentially enabling unlimited spatial gene regulation patterns well beyond those granted by mere gradients.

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.

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.

SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments.

The protein kinase TOUSLED facilitates RNAi in Arabidopsis.

RNA silencing is an evolutionarily conserved mechanism triggered by double-stranded RNA that is processed into 21- to 24-nt small interfering (si)RNA or micro (mi)RNA by RNaseIII-like enzymes called Dicers. Gene regulations by RNA silencing have fundamental implications in a large number of biological processes that include antiviral defense, maintenance of genome integrity and the orchestration of cell fates. Although most generic or core components of the various plant small RNA pathways have been likely identified over the past 15 years, factors involved in RNAi regulation through post-translational modifications are just starting to emerge, mostly through forward genetic studies. A genetic screen designed to identify factors required for RNAi in Arabidopsis identified the serine/threonine protein kinase, TOUSLED (TSL). Mutations in TSL affect exogenous and virus-derived siRNA activity in a manner dependent upon its kinase activity. By contrast, despite their pleiotropic developmental phenotype, tsl mutants show no defect in biogenesis or activity of miRNA or endogenous trans-acting siRNA. These data suggest a possible role for TSL phosphorylation in the specific regulation of exogenous and antiviral RNA silencing in Arabidopsis and identify TSL as an intrinsic regulator of RNA interference.

APC/C-mediated degradation of dsRNA-binding protein 4 (DRB4) involved in RNA silencing.

BACKGROUND: Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed. CONCLUSIONS/SIGNIFICANCE: Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4.

Differential effects of viral silencing suppressors on siRNA and miRNA loading support the existence of two distinct cellular pools of ARGONAUTE1.

Plant viruses encode RNA silencing suppressors (VSRs) to counteract the antiviral RNA silencing response. Based on in-vitro studies, several VSRs were proposed to suppress silencing through direct binding of short-interfering RNAs (siRNAs). Because their expression also frequently hinders endogenous miRNA-mediated regulation and stabilizes labile miRNA* strands, VSRs have been assumed to prevent both siRNA and miRNA loading into their common effector protein, AGO1, through sequestration of small RNA (sRNA) duplexes in vivo. These assumptions, however, have not been formally tested experimentally. Here, we present a systematic in planta analysis comparing the effects of four distinct VSRs in Arabidopsis. While all of the VSRs tested compromised loading of siRNAs into AGO1, only P19 was found to concurrently prevent miRNA loading, consistent with a VSR strategy primarily based on sRNA sequestration. By contrast, we provide multiple lines of evidence that the action of the other VSRs tested is unlikely to entail siRNA sequestration, indicating that in-vitro binding assays and in-vivo miRNA* stabilization are not reliable indicator of VSR action. The contrasted effects of VSRs on siRNA versus miRNA loading into AGO1 also imply the existence of two distinct pools of cellular AGO1 that are specifically loaded by each class of sRNAs. These findings have important implications for our current understanding of RNA silencing and of its suppression in plants.

Isoprenoid biosynthesis is required for miRNA function and affects membrane association of ARGONAUTE 1 in Arabidopsis.

Plant and metazoan microRNAs (miRNAs) guide ARGONAUTE (AGO) protein complexes to regulate expression of complementary RNAs via base pairing. In the plant Arabidopsis thaliana, the main miRNA effector is AGO1, but few other factors required for miRNA activity are known. Here, we isolate the genes defined by the previously described miRNA action deficient (mad) mutants, mad3 and mad4. Both genes encode enzymes involved in isoprenoid biosynthesis. MAD3 encodes 3-hydroxy-3-methylglutaryl CoA reductase (HMG1), which functions in the initial C(5) building block biogenesis that precedes isoprenoid metabolism. HMG1 is a key regulatory enzyme that controls the amounts of isoprenoid end products. MAD4 encodes sterol C-8 isomerase (HYDRA1) that acts downstream in dedicated sterol biosynthesis. Using yeast complementation assays and in planta application of lovastatin, a competitive inhibitor of HMG1, we show that defects in HMG1 catalytic activity are sufficient to inhibit miRNA activity. Many isoprenoid derivatives are indispensable structural and signaling components, and especially sterols are essential membrane constituents. Accordingly, we provide evidence that AGO1 is a peripheral membrane protein. Moreover, specific hypomorphic mutant alleles of AGO1 display compromised membrane association and AGO1-membrane interaction is reduced upon knockdown of HMG1/MAD3. These results suggest a possible basis for the requirement of isoprenoid biosynthesis for the activity of plant miRNAs, and unravel mechanistic features shared with their metazoan counterparts.

An endogenous, systemic RNAi pathway in plants.

Recent work on metazoans has uncovered the existence of an endogenous RNA-silencing pathway that functionally recapitulates the effects of experimental RNA interference (RNAi) used for gene knockdown in organisms such as Caenorhabditis elegans and Drosophila. The endogenous short interfering (si)RNA involved in this pathway are processed by Dicer-like nucleases from genomic loci re-arranged to form extended inverted repeats (IRs) that produce perfect or near-perfect dsRNA molecules. Although such IR loci are commonly detected in plant genomes, their genetics, evolution and potential contribution to plant biology through endogenous silencing have remained largely unexplored. Through an exhaustive analysis performed using Arabidopsis, we provide here evidence that at least two such endogenous IRs are genetically virtually indistinguishable from the transgene constructs commonly used for RNAi in plants. We show how these loci can be useful probes of the cellular mechanism and fluidity of RNA-silencing pathways in plants, and provide evidence that they may arise and disappear on an ecotype scale, show highly cell-specific expression patterns and respond to various stresses. IR loci thus have the potential to act as molecular sensors of the local environments found within distinct ecological plant niches. We further show that the various siRNA size classes produced by at least one of these IR loci are functionally loaded into cognate effector proteins and mediate both post-transcriptional gene silencing and RNA-directed DNA methylation (RdDM) of endogenous as well as exogenous targets. Finally, and as previously reported during plant experimental RNAi, we provide evidence that endogenous IR-derived siRNAs of all size classes are not cell-autonomous and can be transported through graft junctions over long distances, in target tissues where they are functional, at least in mediating RdDM. Collectively, these results define the existence of a bona fide, endogenous and systemic RNAi pathway in plants that may have implications in adaptation, epiallelism and trans-generational memory.

Small RNA duplexes function as mobile silencing signals between plant cells.

In the plant RNA interference (RNAi) pathway, 21-nucleotide duplexes of small interfering RNA (siRNA) are processed from longer double-stranded RNA precursors by the RNaseIII Dicer-like 4 (DCL4). Single-stranded siRNAs then guide Argonaute 1 (AGO1) to execute posttranscriptional silencing of complementary target RNAs. RNAi is not cell-autonomous in higher plants, but the nature of the mobile nucleic acid(s) signal remains unknown. Using cell-specific rescue of DCL4 function and cell-specific inhibition of RNAi movement, we genetically establish that exogenous and endogenous siRNAs, as opposed to their precursor molecules, act as mobile silencing signals between plant cells. We further demonstrate physical movement of mechanically delivered, labeled siRNA duplexes that functionally recapitulate transgenic RNAi spread. Cell-to-cell movement is unlikely to involve AGO1-bound siRNA single strands, but instead likely involves siRNA duplexes.

Molecular dissection of the prototype foamy virus (PFV) RNA 5'-UTR identifies essential elements of a ribosomal shunt.

The prototype foamy virus (PFV) is a nonpathogenic retrovirus that shows promise as a vector for gene transfer. The PFV (pre)genomic RNA starts with a long complex leader that can be folded into an elongated hairpin, suggesting an alternative strategy to cap-dependent linear scanning for translation initiation of the downstream GAG open reading frame (ORF). We found that the PFV leader carries several short ORFs (sORFs), with the three 5'-proximal sORFs located upstream of a structural element. Scanning-inhibitory hairpin insertion analysis suggested a ribosomal shunt mechanism, whereby ribosomes start scanning at the leader 5'-end and initiate at the downstream ORF via bypass of the central leader regions, which are inhibitory for scanning. We show that the efficiency of shunting depends strongly on the stability of the structural element located downstream of either sORFs A/A' or sORF B, and on the translation event at the corresponding 5'-proximal sORF. The PFV shunting strategy mirrors that of Cauliflower mosaic virus in plants; however, in mammals shunting can operate in the presence of a less stable structural element, although it is greatly improved by increasing the number of base pairings. At least one shunt configuration was found in primate FV (pre)genomic RNAs.

Calibrated forceps: a sensitive and reliable tool for pain and analgesia studies.

UNLABELLED: Devices designed for mechanical pain threshold studies are often difficult to implement. The purpose of this study was to investigate a simple tool based on calibrated forceps to induce quantifiable mechanical stimulation in the rat on a linear scale. The most suitable protocol was tested by determining the effects of 3 repetitive measurements on both hind paws, respectively, during long-term (9 days), mid-term (1 day), and short-term (2 hours). Only threshold increase related to weight gain over long-term was observed, suggesting that moderate rat training can be used. The capacity of the device to reveal hyperalgesia was tested in a model of carrageenan-induced inflammation in the hind paw. The hyperalgesia was maximal 6 hours after carrageenan injection and progressively decreased. Similar, although more variable, responses were observed with von Frey filaments. Morphine-induced analgesia resulted in a dose-dependent increase of paw threshold. Tolerance to morphine administrated on a once daily schedule (10 mg/kg) during 5 days was revealed by a significant decrease in analgesia by day 3. Taken together, these results demonstrated accuracy of this device for easy, fast, and reproducible measure of mechanical pain threshold on rat limbs. Moreover, it allows the performance of rat testing with minimal constraint, which reduces data variability. PERSPECTIVE: The calibrated forceps is an easy to use device well-suited to rapidly test mechanical pain threshold with accuracy. It is well-designed for preclinical behavioral screening of noxious or analgesic properties of molecules.