The unique dual targeting of AGO1 by two types of PRMT enzymes promotes phasiRNA loading in Arabidopsis thaliana.

Arginine/R methylation (R-met) of proteins is a widespread post-translational modification (PTM), deposited by a family of protein arginine/R methyl transferase enzymes (PRMT). Regulations by R-met are involved in key biological processes deeply studied in metazoan. Among those, post-transcriptional gene silencing (PTGS) can be regulated by R-met in animals and in plants.

Cell-type-specific control of secondary cell wall formation by Musashi-type translational regulators in .

Deciphering the mechanism of secondary cell wall/SCW formation in plants is key to understanding their development and the molecular basis of biomass recalcitrance. Although transcriptional regulation is essential for SCW formation, little is known about the implication of post-transcriptional mechanisms in this process. Here we report that two RNA-binding proteins homologous to the animal translational regulator Musashi, MSIL2 and MSIL4, function redundantly to control SCW formation in .

Loss of Polycomb proteins CLF and LHP1 leads to excessive RNA degradation in Arabidopsis.

Polycomb-group (PcG) proteins are major chromatin complexes that regulate gene expression, mainly described as repressors keeping genes in a transcriptionally silent state during development. Recent studies have nonetheless suggested that PcG proteins might have additional functions, including targeting active genes or acting independently of gene expression regulation. However, the reasons for the implication of PcG proteins and their associated chromatin marks on active genes are still largely unknown.

A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental gene partitioning in .

Correct 3'end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly arranged stage-specific genes. In this study, we report our findings in of an m6A-dependent 3'end polyadenylation serving as a transcriptional barrier at these .

The mA pathway protects the transcriptome integrity by restricting RNA chimera formation in plants.

Global, segmental, and gene duplication-related processes are driving genome size and complexity in plants. Despite their evolutionary potentials, those processes can also have adverse effects on genome regulation, thus implying the existence of specialized corrective mechanisms. Here, we report that an N6-methyladenosine (mA)-assisted polyadenylation (m-ASP) pathway ensures transcriptome integrity in Efficient m-ASP pathway activity requires the mA methyltransferase-associated factor FIP37 and CPSF30L, an mA reader corresponding to an YT512-B Homology Domain-containing protein (YTHDC)-type domain containing isoform of the 30-kD subunit of cleavage and polyadenylation specificity factor.

UAP56 associates with DRM2 and is localized to chromatin in Arabidopsis.

Repeated sequence expression and transposable element mobilization are tightly controlled by multilayer processes, which include DNA 5'-cytosine methylation. The RNA-directed DNA methylation (RdDM) pathway, which uses siRNAs to guide sequence-specific directed DNA methylation, emerged specifically in plants. RdDM ensures DNA methylation maintenance on asymmetric CHH sites and specifically initiates de novo methylation in all cytosine sequence contexts through the action of DRM DNA methyltransferases, of which DRM2 is the most prominent.

Evidence for ARGONAUTE4-DNA interactions in RNA-directed DNA methylation in plants.

RNA polymerase V (Pol V) long noncoding RNAs (lncRNAs) have been proposed to guide ARGONAUTE4 (AGO4) to chromatin in RNA-directed DNA methylation (RdDM) in plants. Here, we provide evidence, based on laser UV-assisted zero-length cross-linking, for functionally relevant AGO4-DNA interaction at RdDM targets. We further demonstrate that Pol V lncRNAs or the act of their transcription are required to lock Pol V holoenzyme into a stable DNA-bound state that allows AGO4 recruitment via redundant glycine-tryptophan/tryptophan-glycine AGO hook motifs present on both Pol V and its associated factor, SPT5L.

The zinc-finger protein SPT4 interacts with SPT5L/KTF1 and modulates transcriptional silencing in Arabidopsis.

The Arabidopsis multidomain protein SPT5L/KTF1 (which has similarity to the transcript elongation factor SPT5) associates with RNA polymerase V (RNAPV) and is an accessory factor in RNA-directed DNA methylation. The zinc-finger protein SPT4 was found to interact with SPT5L (and SPT5) both in vivo and in vitro. Here, we show that plants depleted of SPT4 relative to wild type display reduced DNA methylation and the locus specificity is shared with SPT5L, suggesting a cooperation of SPT4 and SPT5L.

Ago hook and RNA helicase motifs underpin dual roles for SDE3 in antiviral defense and silencing of nonconserved intergenic regions.

In Arabidopsis thaliana, the putative RNA-helicase SDE3 assists posttranscriptional-gene-silencing (PTGS) amplification by RNA-dependent-RNA-polymerase-6 (RDR6). SDE3 homologs in Drosophila, worm and human contribute to silence viruses, transposons or recently duplicated genes but the underlying mechanisms remain largely unknown. Here, we demonstrate that SDE3 is present with the PTGS effectors AGO1 and AGO2 in higher-order protein complexes owing to a specialized GW-repeat-containing C-terminal domain.

NERD, a plant-specific GW protein, defines an additional RNAi-dependent chromatin-based pathway in Arabidopsis.

In Arabidopsis, transcriptional gene silencing (TGS) can be triggered by 24 nt small-interfering RNAs (siRNAs) through the RNA-directed DNA methylation (RdDM) pathway. By functional analysis of NERD, a GW repeat- and PHD finger-containing protein, we demonstrate that Arabidopsis harbors a second siRNA-dependent DNA methylation pathway targeting a subset of nonconserved genomic loci. The activity of the NERD-dependent pathway differs from RdDM by the fact that it relies both on silencing-related factors previously implicated only in posttranscriptional gene silencing (PTGS), including RNA-DEPENDENT RNA POLYMERASE1/6 and ARGONAUTE2, and most likely on 21 nt siRNAs.

Human prion protein binds Argonaute and promotes accumulation of microRNA effector complexes.

Despite intense research in the context of neurodegenerative diseases associated with its misfolding, the endogenous human prion protein PrP(C) (or PRNP) has poorly understood physiological functions. Whereas most PrP(C) is exposed to the extracellular environment, conserved domains result in transmembrane forms of PrP(C) that traffic in the endolysosomal system and are linked to inherited and infectious neuropathologies. One transmembrane PrP(C) variant orients the N-terminal 'octarepeat' domain into the cytoplasm.

Taking RISCs with Ago hookers.

Argonautes are central and common components of crucial effectors of RNA silencing pathways. Although earlier steps in these pathways, such as small RNA biogenesis and their loading into AGO, have been quite well described, our knowledge on regulation of the action of AGO and their partners is still poor. Recent breakthroughs have highlighted the existence in many eukaryotes of an evolutionarily conserved motif, the Ago-hook, in factors implicated in AGO action.

A plant-specific transcription factor IIB-related protein, pBRP2, is involved in endosperm growth control.

General transcription factor IIB (TFIIB) and TFIIB-related factor (BRF), are conserved RNA polymerase II/III (RNAPII/III) selectivity factors that are involved in polymerase recruitment and transcription initiation in eukaryotes. Recent findings have shown that plants have evolved a third type of B-factor, plant-specific TFIIB-related protein 1 (pBRP1), which seems to be involved in RNAPI transcription. Here, we extend the repertoire of B-factors in plants by reporting the characterization of a novel TFIIB-related protein, plant-specific TFIIB-related protein 2 (pBRP2), which is found to date only in the Brassicacea family.

Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein.

In plants and invertebrates, viral-derived siRNAs processed by the RNaseIII Dicer guide Argonaute (AGO) proteins as part of antiviral RNA-induced silencing complexes (RISC). As a counterdefense, viruses produce suppressor proteins (VSRs) that inhibit the host silencing machinery, but their mechanisms of action and cellular targets remain largely unknown. Here, we show that the Turnip crinckle virus (TCV) capsid, the P38 protein, acts as a homodimer, or multiples thereof, to mimic host-encoded glycine/tryptophane (GW)-containing proteins normally required for RISC assembly/function in diverse organisms.

An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation.

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1.

Genome-wide computational identification of WG/GW Argonaute-binding proteins in Arabidopsis.

Domains in Arabidopsis proteins NRPE1 and SPT5-like, composed almost exclusively of repeated motifs in which only WG or GW sequences and an overall amino-acid preference are conserved, have been experimentally shown to bind multiple molecules of Argonaute (AGO) protein(s). Domain swapping between the WG/GW domains of NRPE1 and the human protein GW182 showed a conserved function. As classical sequence alignment methods are poorly-adapted to detect such weakly-conserved motifs, we have developed a tool to carry out a systematic analysis to identify genes potentially encoding AGO-binding GW/WG proteins.

Plant-specific multisubunit RNA polymerase in gene silencing.

In recent years, a major breakthrough in the study of epigenetic silencing in eukaryotes came with the discovery that the RNA-interference pathway (RNAi) is generally implicated in heterochromatin assembly and gene silencing. An important and paradoxical feature of the RNAi-mediated heterochromatin pathways is their requirement for some form of transcription. In fission yeast, Schizosaccharomyces pombe, centromeric siRNAs have been shown to derive from chromatin-bound nascent transcripts produced by RNA polymerase II (PolII) at the site of heterochromatin formation.

RNA-directed DNA methylation requires an AGO4-interacting member of the SPT5 elongation factor family.

Recent studies have identified a conserved WG/GW-containing motif, known as the Argonaute (AGO) hook, which is involved in the recruitment of AGOs to distinct components of the eukaryotic RNA silencing pathways. By using this motif as a model to detect new components in plant RNA silencing pathways, we identified SPT5-like, a plant-specific AGO4-interacting member of the nuclear SPT5 (Suppressor of Ty insertion 5) RNA polymerase (RNAP) elongation factor family that is characterized by the presence of a carboxy-terminal extension with more than 40 WG/GW motifs. Knockout SPT5-like mutants show a decrease in the accumulation of several 24-nt RNAs and hypomethylation at different loci revealing an implication in RNA-directed DNA methylation (RdDM).

PolV(PolIVb) function in RNA-directed DNA methylation requires the conserved active site and an additional plant-specific subunit.

Two forms of a plant-specific RNA polymerase (Pol), PolIV(PolIVa) and PolV(PolIVb), currently defined by their respective largest subunits [NRPD1(NRPD1a) and NRPE1(NRPD1b)], have been implicated in the production and activity of 24-nt small RNAs (sRNAs) in RNA-directed DNA methylation (RdDM). Prevailing models support the view that PolIV(PolIVa) plays an upstream role in RdDM by producing the 24-nt sRNAs, whereas PolV(PolIVb) would act downstream at a structural rather than an enzymatic level to reinforce sRNA production by PolIV(PolIVa) and mediate DNA methylation. However, the composition and mechanism of action of PolIV(PolIVa)/PolV(PolIVb) remain unclear.

The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis.

Proteins containing the forkhead-associated domain (FHA) are known to act in biological processes such as DNA damage repair, protein degradation, and signal transduction. Here we report that DAWDLE (DDL), an FHA domain-containing protein in Arabidopsis, acts in the biogenesis of miRNAs and endogenous siRNAs. Unlike mutants of genes known to participate in the processing of miRNA precursors, such as dcl1, hyponastic leaves1, and serrate, ddl mutants show reduced levels of pri-miRNAs as well as mature miRNAs.

Dynamic regulation of ARGONAUTE4 within multiple nuclear bodies in Arabidopsis thaliana.

DNA methylation directed by 24-nucleotide small RNAs involves the small RNA-binding protein ARGONAUTE4 (AGO4), and it was previously shown that AGO4 localizes to nucleolus-adjacent Cajal bodies, sites of snRNP complex maturation. Here we demonstrate that AGO4 also localizes to a second class of nuclear bodies, called AB-bodies, which are found immediately adjacent to condensed 45S ribosomal DNA (rDNA) sequences. AB-bodies also contain other proteins involved in RNA-directed DNA methylation including NRPD1b (a subunit of the RNA Polymerase IV complex, RNA PolIV), NRPD2 (a second subunit of this complex), and the DNA methyltransferase DRM2.

Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components.

Two forms of RNA Polymerase IV (PolIVa/PolIVb) have been implicated in RNA-directed DNA methylation (RdDM) in Arabidopsis. Prevailing models imply a distinct function for PolIVb by association of Argonaute4 (AGO4) with the C-terminal domain (CTD) of its largest subunit NRPD1b. Here we show that the extended CTD of NRPD1b-type proteins exhibits conserved Argonaute-binding capacity through a WG/GW-rich region that functionally distinguishes Pol IVb from Pol IVa, and that is essential for RdDM.

Knock-out of the magnesium protoporphyrin IX methyltransferase gene in Arabidopsis. Effects on chloroplast development and on chloroplast-to-nucleus signaling.

Protoporphyrin IX is the last common intermediate between the heme and chlorophyll biosynthesis pathways. The addition of magnesium directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the magnesium chelatase and is a highly regulated process.