Hervé Vaucheret

Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants.

Eukaryotic RNA quality control (RQC) uses both endonucleolytic and exonucleolytic degradation to eliminate dysfunctional RNAs. In addition, endogenous and exogenous RNAs are degraded through post-transcriptional gene silencing (PTGS), which is triggered by the production of double-stranded (ds)RNAs and proceeds through short-interfering (si)RNA-directed ARGONAUTE-mediated endonucleolytic cleavage. Compromising cytoplasmic or nuclear 5′–3′ exoribonuclease function enhances sense-transgene (S)-PTGS in Arabidopsis, suggesting that these pathways compete for similar RNA substrates. Here, we show that impairing nonsense-mediated decay, deadenylation or exosome activity enhanced S-PTGS, which requires host RNA-dependent RNA polymerase 6 (RDR6/SGS2/SDE1) and SUPPRESSOR OF GENE SILENCING 3 (SGS3) for the transformation of single-stranded RNA into dsRNA to trigger PTGS. However, these RQC mutations had no effect on inverted-repeat–PTGS, which directly produces hairpin dsRNA through transcription. Moreover, we show that these RQC factors are nuclear and cytoplasmic and are found in two RNA degradation foci in the cytoplasm: siRNA-bodies and processing-bodies. We propose a model of single-stranded RNA tug-of-war between RQC and S-PTGS that ensures the correct partitioning of RNA substrates among these RNA degradation pathways.

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.

Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses

Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant–virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.

Arabidopsis RNASE THREE LIKE2 Modulates the Expression of Protein-Coding Genes via 24-Nucleotide Small Interfering RNA-Directed DNA Methylation

The Arabidopsis RNaseIII enzyme RTL2 modulates the 24-nucleotide small interfering RNA repertoire and contributes to the regulation of protein-coding gene expression. RNaseIII enzymes catalyze the cleavage of double-stranded RNA (dsRNA) and have diverse functions in RNA maturation. Arabidopsis thaliana RNASE THREE LIKE2 (RTL2), which carries one RNaseIII and two dsRNA binding (DRB) domains, is a unique Arabidopsis RNaseIII enzyme resembling the budding yeast small interfering RNA (siRNA)-producing Dcr1 enzyme. Here, we show that RTL2 modulates the production of a subset of small RNAs and that this activity depends on both its RNaseIII and DRB domains. However, the mode of action of RTL2 differs from that of Dcr1. Whereas Dcr1 directly cleaves dsRNAs into 23-nucleotide siRNAs, RTL2 likely cleaves dsRNAs into longer molecules, which are subsequently processed into small RNAs by the DICER-LIKE enzymes. Depending on the dsRNA considered, RTL2-mediated maturation either improves (RTL2-dependent loci) or reduces (RTL2-sensitive loci) the production of small RNAs. Because the vast majority of RTL2-regulated loci correspond to transposons and intergenic regions producing 24-nucleotide siRNAs that guide DNA methylation, RTL2 depletion modifies DNA methylation in these regions. Nevertheless, 13% of RTL2-regulated loci correspond to protein-coding genes. We show that changes in 24-nucleotide siRNA levels also affect DNA methylation levels at such loci and inversely correlate with mRNA steady state levels, thus implicating RTL2 in the regulation of protein-coding gene expression.

RNA silencing is resistant to low-temperature in grapevine.

RNA silencing is a natural defence mechanism against viruses in plants, and transgenes expressing viral RNA-derived sequences were previously shown to confer silencing-based enhanced resistance against the cognate virus in several species. However, RNA silencing was shown to dysfunction at low temperatures in several species, questioning the relevance of this strategy in perennial plants such as grapevines, which are often exposed to low temperatures during the winter season. Here, we show that inverted-repeat (IR) constructs trigger a highly efficient silencing reaction in all somatic tissues in grapevines. Similarly to other plant species, IR-derived siRNAs trigger production of secondary transitive siRNAs. However, and in sharp contrast to other species tested to date where RNA silencing is hindered at low temperature, this process remained active in grapevine cultivated at 4°C. Consistently, siRNA levels remained steady in grapevines cultivated between 26°C and 4°C, whereas they are severely decreased in Arabidopsis grown at 15°C and almost undetectable at 4°C. Altogether, these results demonstrate that RNA silencing operates in grapevine in a conserved manner but is resistant to far lower temperatures than ever described in other species.

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.

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

SmD1, a conserved component of the small nuclear ribonucleoprotein complex, participates in splicing and in the partitioning of aberrant RNAs between RNA quality control and RNA silencing pathways. 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 siRNA suppressor RTL1 is redox-regulated through glutathionylation of a conserved cysteine in the double-stranded-RNA-binding domain

Abstract RNase III enzymes cleave double stranded (ds)RNA. This is an essential step for regulating the processing of mRNA, rRNA, snoRNA and other small RNAs, including siRNA and miRNA. Arabidopsis thaliana encodes nine RNase III: four DICER-LIKE (DCL) and five RNASE THREE LIKE (RTL). To better understand the molecular functions of RNase III in plants we developed a biochemical assay using RTL1 as a model. We show that RTL1 does not degrade dsRNA randomly, but recognizes specific duplex sequences to direct accurate cleavage. Furthermore, we demonstrate that RNase III and dsRNA binding domains (dsRBD) are both required for dsRNA cleavage. Interestingly, the four DCL and the three RTL that carry dsRBD share a conserved cysteine (C230 in Arabidopsis RTL1) in their dsRBD. C230 is essential for RTL1 and DCL1 activities and is subjected to post-transcriptional modification. Indeed, under oxidizing conditions, glutathionylation of C230 inhibits RTL1 cleavage activity in a reversible manner involving glutaredoxins. We conclude that the redox state of the dsRBD ensures a fine-tune regulation of dsRNA processing by plant RNase III.

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

The AGO1 DUF1785, which is a target of the polerovirus F-box P0 protein, is required for unwinding perfectly matched siRNA duplexes and its mutation abolishes phasiRNA production and transgene S-PTGS 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.

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.