Synergistic action of the Arabidopsis spliceosome components PRP39a and SmD1b in promoting posttranscriptional transgene silencing.

Besides regulating splicing, the conserved spliceosome component SmD1 (Small nuclear ribonucleoprotein D1)b promotes posttranscriptional silencing of sense transgenes (S-PTGS [post-transcriptional genesilencing]). Here, we show that the conserved spliceosome component PRP39 (Pre-mRNA-processing factor 39)a also plays a role in S-PTGS in Arabidopsis thaliana. However, PRP39a and SmD1b actions appear distinct in both splicing and S-PTGS. Indeed, RNAseq-based analysis of expression level and alternative splicing in prp39a and smd1b mutants identified different sets of deregulated transcripts and noncoding RNAs. Moreover, double mutant analyses involving prp39a or smd1b and RNA quality control (RQC) mutants revealed distinct genetic interactions for SmD1b and PRP39a with nuclear RQC machineries, suggesting nonredundant roles in the RQC/PTGS interplay. Supporting this hypothesis, a prp39a smd1b double mutant exhibited enhanced suppression of S-PTGS compared to the single mutants. Because the prp39a and smd1b mutants (i) showed no major changes in the expression of PTGS or RQC components or in small RNA production and (ii) do not alter PTGS triggered by inverted-repeat transgenes directly producing dsRNA (IR-PTGS), PRP39a, and SmD1b appear to synergistically promote a step specific to S-PTGS. We propose that, independently from their specific roles in splicing, PRP39a and SmD1b limit 3'-to-5' and/or 5'-to-3' degradation of transgene-derived aberrant RNAs in the nucleus, thus favoring the export of aberrant RNAs to the cytoplasm where their conversion into double-stranded RNA (dsRNA) initiates S-PTGS.

The viral F-box protein P0 induces an ER-derived autophagy degradation pathway for the clearance of membrane-bound AGO1.

RNA silencing is a major antiviral defense mechanism in plants and invertebrates. Plant ARGONAUTE1 (AGO1) is pivotal in RNA silencing, and hence is a major target for counteracting viral suppressors of RNA-silencing proteins (VSRs). P0 from Turnip yellows virus (TuYV) is a VSR that was previously shown to trigger AGO1 degradation via an autophagy-like process. However, the identity of host proteins involved and the cellular site at which AGO1 and P0 interact were unknown. Here we report that P0 and AGO1 associate on the endoplasmic reticulum (ER), resulting in their loading into ER-associated vesicles that are mobilized to the vacuole in an ATG5- and ATG7-dependent manner. We further identified ATG8-Interacting proteins 1 and 2 (ATI1 and ATI2) as proteins that associate with P0 and interact with AGO1 on the ER up to the vacuole. Notably, ATI1 and ATI2 belong to an endogenous degradation pathway of ER-associated AGO1 that is significantly induced following P0 expression. Accordingly, ATI1 and ATI2 deficiency causes a significant increase in posttranscriptional gene silencing (PTGS) activity. Collectively, we identify ATI1 and ATI2 as components of an ER-associated AGO1 turnover and proper PTGS maintenance and further show how the VSR P0 manipulates this pathway.

The root-knot nematode effector MiEFF18 interacts with the plant core spliceosomal protein SmD1 required for giant cell formation.

The root-knot nematode Meloidogyne incognita secretes specific effectors (MiEFF) and induces the redifferentiation of plant root cells into enlarged multinucleate feeding 'giant cells' essential for nematode development. Immunolocalizations revealed the presence of the MiEFF18 protein in the salivary glands of M. incognita juveniles. In planta, MiEFF18 localizes to the nuclei of giant cells demonstrating its secretion during plant-nematode interactions. A yeast two-hybrid approach identified the nuclear ribonucleoprotein SmD1 as a MiEFF18 partner in tomato and Arabidopsis. SmD1 is an essential component of the spliceosome, a complex involved in pre-mRNA splicing and alternative splicing. RNA-seq analyses of Arabidopsis roots ectopically expressing MiEFF18 or partially impaired in SmD1 function (smd1b mutant) revealed the contribution of the effector and its target to alternative splicing and proteome diversity. The comparison with Arabidopsis galls data showed that MiEFF18 modifies the expression of genes important for giant cell ontogenesis, indicating that MiEFF18 modulates SmD1 functions to facilitate giant cell formation. Finally, Arabidopsis smd1b mutants exhibited less susceptibility to M. incognita infection, and the giant cells formed on these mutants displayed developmental defects, suggesting that SmD1 plays an important role in the formation of giant cells and is required for successful nematode infection.

Post-transcriptional gene silencing triggers dispensable DNA methylation in gene body in Arabidopsis.

Spontaneous post-transcriptional silencing of sense transgenes (S-PTGS) is established in each generation and is accompanied by DNA methylation, but the pathway of PTGS-dependent DNA methylation is unknown and so is its role. Here we show that CHH and CHG methylation coincides spatially and temporally with RDR6-dependent products derived from the central and 3' regions of the coding sequence, and requires the components of the RNA-directed DNA methylation (RdDM) pathway NRPE1, DRD1 and DRM2, but not CLSY1, NRPD1, RDR2 or DCL3, suggesting that RDR6-dependent products, namely long dsRNAs and/or siRNAs, trigger PTGS-dependent DNA methylation. Nevertheless, none of these RdDM components are required to establish S-PTGS or produce a systemic silencing signal. Moreover, preventing de novo DNA methylation in non-silenced transgenic tissues grafted onto homologous silenced tissues does not inhibit the triggering of PTGS. Overall, these data indicate that gene body DNA methylation is a consequence, not a cause, of PTGS, and rule out the hypothesis that a PTGS-associated DNA methylation signal is transmitted independent of a PTGS signal.

The Nuclear Ribonucleoprotein SmD1 Interplays with Splicing, RNA Quality Control, and Posttranscriptional Gene Silencing in Arabidopsis.

RNA quality control (RQC) eliminates aberrant RNAs based on their atypical structure, whereas posttranscriptional gene silencing (PTGS) eliminates both aberrant and functional RNAs through the sequence-specific action of short interfering RNAs (siRNAs). The Arabidopsis thaliana mutant smd1b was identified in a genetic screen for PTGS deficiency, revealing the involvement of SmD1, a component of the Smith (Sm) complex, in PTGS. The smd1a and smd1b single mutants are viable, but the smd1a smd1b double mutant is embryo-lethal, indicating that SmD1 function is essential. SmD1b resides in nucleoli and nucleoplasmic speckles, colocalizing with the splicing-related factor SR34. Consistent with this, the smd1b mutant exhibits intron retention at certain endogenous mRNAs. SmD1 binds to RNAs transcribed from silenced transgenes but not nonsilenced ones, indicating a direct role in PTGS. Yet, mutations in the RQC factors UPFRAMESHIFT3, EXORIBONUCLEASE2 (XRN2), XRN3, and XRN4 restore PTGS in smd1b, indicating that SmD1 is not essential for but rather facilitates PTGS. Moreover, the smd1b mtr4 double mutant is embryo-lethal, suggesting that SmD1 is essential for mRNA TRANSPORT REGULATOR4-dependent RQC. These results indicate that SmD1 interplays with splicing, RQC, and PTGS. We propose that SmD1 facilitates PTGS by protecting transgene-derived aberrant RNAs from degradation by RQC in the nucleus, allowing sufficient amounts to enter cytoplasmic siRNA bodies to activate PTGS.

The Zinc-Finger Protein SOP1 Is Required for a Subset of the Nuclear Exosome Functions in Arabidopsis.

Correct gene expression requires tight RNA quality control both at transcriptional and post-transcriptional levels. Using a splicing-defective allele of PASTICCINO2 (PAS2), a gene essential for plant development, we isolated suppressor mutations modifying pas2-1 mRNA profiles and restoring wild-type growth. Three suppressor of pas2 (sop) mutations modified the degradation of mis-spliced pas2-1 mRNA species, allowing the synthesis of a functional protein. Cloning of the suppressor mutations identified the core subunit of the exosome SOP2/RRP4, the exosome nucleoplasmic cofactor SOP3/HEN2 and a novel zinc-finger protein SOP1 that colocalizes with HEN2 in nucleoplasmic foci. The three SOP proteins counteract post-transcriptional (trans)gene silencing (PTGS), which suggests that they all act in RNA quality control. In addition, sop1 mutants accumulate some, but not all of the misprocessed mRNAs and other types of RNAs that are observed in exosome mutants. Taken together, our data show that SOP1 is a new component of nuclear RNA surveillance that is required for the degradation of a specific subset of nuclear exosome targets.

sgs1: a neomorphic nac52 allele impairing post-transcriptional gene silencing through SGS3 downregulation.

Post-transcriptional gene silencing (PTGS) is a defense mechanism that targets invading nucleic acids from endogenous (transposons) or exogenous (pathogens, transgenes) sources. Genetic screens based on the reactivation of silenced transgenes have long been used to identify cellular components and regulators of PTGS. Here we show that the first isolated PTGS-deficient mutant, sgs1, is impaired in the transcription factor NAC52. This mutant exhibits striking similarities to a mutant impaired in the H3K4me3 demethylase JMJ14 isolated from the same genetic screen. These similarities include increased transgene promoter DNA methylation, reduced H3K4me3 and H3K36me3 levels, reduced PolII occupancy and reduced transgene mRNA accumulation. It is likely that increased DNA methylation is the cause of reduced transcription because the effect of jmj14 and sgs1 on transgene transcription is suppressed by drm2, a mutation that compromises de novo DNA methylation, suggesting that the JMJ14-NAC52 module promotes transgene transcription by preventing DNA methylation. Remarkably, sgs1 has a stronger effect than jmj14 and nac52 null alleles on PTGS systems requiring siRNA amplification, and this is due to reduced SGS3 mRNA levels in sgs1. Given that the sgs1 mutation changes a conserved amino acid of the NAC proteins involved in homodimerization, we propose that sgs1 corresponds to a neomorphic nac52 allele encoding a mutant protein that lacks wild-type NAC52 activity but promotes SGS3 downregulation. Together, these results indicate that impairment of PTGS in sgs1 is due to its dual effect on transgene transcription and SGS3 transcription, thus compromising siRNA amplification.

The RNA helicases AtMTR4 and HEN2 target specific subsets of nuclear transcripts for degradation by the nuclear exosome in Arabidopsis thaliana.

The RNA exosome is the major 3'-5' RNA degradation machine of eukaryotic cells and participates in processing, surveillance and turnover of both nuclear and cytoplasmic RNA. In both yeast and human, all nuclear functions of the exosome require the RNA helicase MTR4. We show that the Arabidopsis core exosome can associate with two related RNA helicases, AtMTR4 and HEN2. Reciprocal co-immunoprecipitation shows that each of the RNA helicases co-purifies with the exosome core complex and with distinct sets of specific proteins. While AtMTR4 is a predominantly nucleolar protein, HEN2 is located in the nucleoplasm and appears to be excluded from nucleoli. We have previously shown that the major role of AtMTR4 is the degradation of rRNA precursors and rRNA maturation by-products. Here, we demonstrate that HEN2 is involved in the degradation of a large number of polyadenylated nuclear exosome substrates such as snoRNA and miRNA precursors, incompletely spliced mRNAs, and spurious transcripts produced from pseudogenes and intergenic regions. Only a weak accumulation of these exosome substrate targets is observed in mtr4 mutants, suggesting that MTR4 can contribute, but plays rather a minor role for the degradation of non-ribosomal RNAs and cryptic transcripts in Arabidopsis. Consistently, transgene post-transcriptional gene silencing (PTGS) is marginally affected in mtr4 mutants, but increased in hen2 mutants, suggesting that it is mostly the nucleoplasmic exosome that degrades aberrant transgene RNAs to limit their entry in the PTGS pathway. Interestingly, HEN2 is conserved throughout green algae, mosses and land plants but absent from metazoans and other eukaryotic lineages. Our data indicate that, in contrast to human and yeast, plants have two functionally specialized RNA helicases that assist the exosome in the degradation of specific nucleolar and nucleoplasmic RNA populations, respectively.

Respective contributions of Arabidopsis DCL2 and DCL4 to RNA silencing.

Dicer proteins are central to the different mechanisms involving RNA interference. Plants have evolved multiple DICER-LIKE (DCL) copies, thus enabling functional diversification. In Arabidopsis, DCL2 and DCL4 process double-stranded RNA into 22 and 21 nucleotide small interfering (si)RNAs, respectively, and have overlapping functions with regards to virus and transgene silencing. Nonetheless, some studies have reported that dcl2 or dcl4 single mutations are sometimes sufficient to hinder silencing. To better dissect the role of DCL2 and DCL4, we analyzed silencing kinetics and efficiencies using different transgenic systems in single and double mutant backgrounds. The results indicate that DCL2 stimulates transitivity and secondary siRNA production through DCL4 while being sufficient for silencing on its own. Notably, silencing of 35S-driven transgenes functions more efficiently in dcl4 mutants, indicating that DCL4 mostly obscures DCL2 in wild-type plants. Nonetheless, in a dcl4 mutant compromised in phloem-originating silencing, ectopically expressed DCL2 allows restoration of silencing, suggesting that DCL2 is not, or poorly, expressed in phloem. Remarkably, this ectopic DCL2 contribution to phloem-originating silencing is dependent on the activity of RNA-DEPENDENT RNA POLYMERASE6. These results indicate that, despite differences in the silencing activity of their small RNA products, DCL2 and DCL4 mostly act redundantly yet hierarchically when present simultaneously.

Second-Site Mutagenesis of a Hypomorphic argonaute1 Allele Identifies SUPERKILLER3 as an Endogenous Suppressor of Transgene Posttranscriptional Gene Silencing.

Second-site mutagenesis was performed on the argonaute1-33 (ago1-33) hypomorphic mutant, which exhibits reduced sense transgene posttranscriptional gene silencing (S-PTGS). Mutations in FIERY1, a positive regulator of the cytoplasmic 5'-to-3' EXORIBONUCLEASE4 (XRN4), and in SUPERKILLER3 (SKI3), a member of the SKI complex that threads RNAs directly to the 3'-to-5' exoribonuclease of the cytoplasmic exosome, compensated AGO1 partial deficiency and restored S-PTGS with 100% efficiency. Moreover, xrn4 and ski3 single mutations provoked the entry of nonsilenced transgenes into S-PTGS and enhanced S-PTGS on partially silenced transgenes, indicating that cytoplasmic 5'-to-3' and 3'-to-5' RNA degradation generally counteract S-PTGS, likely by reducing the amount of transgene aberrant RNAs that are used by the S-PTGS pathway to build up small interfering RNAs that guide transgene RNA cleavage by AGO1. Constructs generating improperly terminated transgene messenger RNAs (mRNAs) were not more sensitive to ski3 or xrn4 than regular constructs, suggesting that improperly terminated transgene mRNAs not only are degraded from both the 3' end but also from the 5' end, likely after decapping. The facts that impairment of either 5'-to-3' or 3'-to-5' RNA degradation is sufficient to provoke the entry of transgene RNA into the S-PTGS pathway, whereas simultaneous impairment of both pathways is necessary to provoke the entry of endogenous mRNA into the S-PTGS pathway, suggest poor RNA quality upon the transcription of transgenes integrated at random genomic locations.

Mutations in the Arabidopsis H3K4me2/3 demethylase JMJ14 suppress posttranscriptional gene silencing by decreasing transgene transcription.

Posttranscriptional gene silencing (PTGS) mediated by sense transgenes (S-PTGS) results in RNA degradation and DNA methylation of the transcribed region. Through a forward genetic screen, a mutant defective in the Histone3 Lysine4 di/trimethyl (H3K4me2/3) demethylase Jumonji-C (JmjC) domain-containing protein14 (JMJ14) was identified. This mutant reactivates various transgenes silenced by S-PTGS and shows reduced Histone3 Lysine9 Lysine14 acetylation (H3K9K14Ac) levels, reduced polymerase II occupancy, reduced transgene transcription, and increased DNA methylation in the promoter region, consistent with the hypothesis that high levels of transcription are required to trigger S-PTGS. The jmj14 mutation also reduces the expression of transgenes that do not trigger S-PTGS. Moreover, expression of transgenes that undergo S-PTGS in a wild-type background is reduced in jmj14 sgs3 double mutants compared with PTGS-deficient sgs3 mutants, indicating that JMJ14 is required for high levels of transcription in a PTGS-independent manner. Whereas endogenous loci regulated by JMJ14 exhibit increased H3K4me2 and H3K4me3 levels in the jmj14 mutant, transgene loci exhibit unchanged H3K4me2 and decreased H3K4me3 levels. Because jmj14 mutations impair PTGS of transgenes expressed under various plant or viral promoters, we hypothesize that JMJ14 demethylation activity is prevented by antagonistic epigenetic marks specifically imposed at transgene loci. Removing JMJ14 likely allows other H3K4 demethylases encoded by the Arabidopsis thaliana genome to act on transgenes and reduce transcription levels, thus preventing the triggering of S-PTGS.

The pea TCP transcription factor PsBRC1 acts downstream of Strigolactones to control shoot branching.

The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated. The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application. The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes. The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine. The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth. However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth. In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant. Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1. This phenotype indicates a localized regulation of branching at these nodes specific to pea.

The miRNA pathway limits AGO1 availability during siRNA-mediated PTGS defense against exogenous RNA.

In plants, most microRNAs (miRNAs) and several endogenous small interfering RNAs (siRNAs) bind to ARGONAUTE1 (AGO1) to regulate the expression of endogenous genes through post-transcriptional gene silencing (PTGS). AGO1 also participates in a siRNA-mediated PTGS defense response that thwarts exogenous RNA deriving from viruses and transgenes. Here, we reveal that plants supporting transgene PTGS exhibit increased levels of AGO1 protein. Moreover, increasing AGO1 levels either by mutating miRNA pathway components or, more specifically, by impairing miR168-directed regulation of AGO1 mRNA leads to increased PTGS efficiency, indicating that the miRNA pathway dampens the efficiency of PTGS, likely by limiting the availability of AGO1. We propose that during the transgene PTGS initiation phase, transgene siRNAs and endogenous siRNAs and miRNA compete to bind to AGO1, leading to a transient reduction in AGO1-miR168 complexes and a decline in AGO1 mRNA cleavage. The concomitant increase in AGO1 protein levels would facilitate the formation of AGO1-transgene siRNA complexes and the entry into the PTGS amplification phase. We suggest that the miRNA pathway imposes an important limitation on PTGS efficiency, which could help protect endogenous mRNAs from being routinely targeted by PTGS.

The Arabidopsis F-box protein FBW2 targets AGO1 for degradation to prevent spurious loading of illegitimate small RNA.

RNA silencing is a conserved mechanism in eukaryotes involved in development and defense against viruses. In plants, ARGONAUTE1 (AGO1) protein plays a central role in both microRNA- and small interfering RNA-directed silencing, and its expression is regulated at multiple levels. Here, we report that the F-box protein FBW2 assembles an SCF complex that selectively targets for proteolysis AGO1 when it is unloaded and mutated. Although FBW2 loss of function does not lead to strong growth or developmental defects, it significantly increases RNA-silencing activity. Interestingly, under conditions in which small-RNA accumulation is affected, the failure to degrade AGO1 in fbw2 mutants becomes more deleterious for the plant. Accordingly, the non-degradable AGO1 protein assembles high-molecular-weight complexes and binds illegitimate small RNA, leading to off-target cleavage. Therefore, control of AGO1 homeostasis by FBW2 plays an important role in quality control of RNA silencing.

RDR2 partially antagonizes the production of RDR6-dependent siRNA in sense transgene-mediated PTGS.

BACKGROUND: RNA-DEPENDENT RNA POLYMERASE6 (RDR6) and SUPPRESSOR of GENE SILENCING 3 (SGS3) are required for DNA methylation and post-transcriptional gene silencing (PTGS) mediated by 21-nt siRNAs produced by sense transgenes (S-PTGS). In contrast, RDR2, but not RDR6, is required for DNA methylation and TGS mediated by 24-nt siRNAs, and for cell-to-cell spreading of IR-PTGS mediated by 21-nt siRNAs produced by inverted repeat transgenes under the control of a phloem-specific promoter. PRINCIPAL FINDINGS: In this study, we examined the role of RDR2 and RDR6 in S-PTGS. Unlike RDR6, RDR2 is not required for DNA methylation of transgenes subjected to S-PTGS. RDR6 is essential for the production of siRNAs by transgenes subjected to S-PTGS, but RDR2 also contributes to the production of transgene siRNAs when RDR6 is present because rdr2 mutations reduce transgene siRNA accumulation. However, the siRNAs produced via RDR2 likely are counteractive in wildtype plants because impairement of RDR2 increases S-PTGS efficiency at a transgenic locus that triggers limited silencing, and accelerates S-PTGS at a transgenic locus that triggers efficient silencing. CONCLUSIONS/SIGNIFICANCE: These results suggest that RDR2 and RDR6 compete for RNA substrates produced by transgenes subjected to S-PTGS. RDR2 partially antagonizes RDR6 because RDR2 action likely results in the production of counteractive siRNA. As a result, S-PTGS efficiency is increased in rdr2 mutants.