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.     

CRISPR/Cas9-targeted mutagenesis of TaDCL4, TaDCL5 and TaRDR6 induces male sterility in common wheat

Phased, small interfering RNAs (phasiRNAs) are important for plant anther development, especially for male sterility. PhasiRNA biogenesis is dependent on genes like RNA polymerase 6 (RDR6), DICER-LIKE 4 (DCL4), or DCL5 to produce 21- or 24 nucleotide (nt) double-strand small RNAs. Here, we generated mutants of DCL4, DCL5 and RDR6 using CRISPR/Cas9 system and studied their effects on plant reproductive development and phasiRNA production in wheat. We found that RDR6 mutation caused sever consequence throughout plant development starting from seed germination and the dcl4 mutants grew weaker with thorough male sterility, while dcl5 plants developed normally but exhibited male sterility. Correspondingly, DCL4 and DCL5, respectively, specified 21- and 24-nt phasiRNA biogenesis, while RDR6 contributed to both. Also, the three key genes evolved differently in wheat, with TaDCL5-A/B becoming non-functioning and TaRDR6-A being lost after polyploidization. Furthermore, we found that PHAS genes (phasiRNA precursors) identified via phasiRNAs diverged rapidly among sub-genomes of polyploid wheat. Despite no similarity being found among phasiRNAs of grasses, their targets were enriched for similar biological functions. In light of the important roles of phasiRNA pathways in gametophyte development, genetic dissection of the function of key genes may help generate male sterile lines suitable for hybrid wheat breeding.     

An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs

Plants contain more DICER-LIKE (DCL) enzymes and double-stranded RNA binding (DRB) proteins than other eukaryotes, resulting in increased small RNA network complexities. Analyses of single, double, triple and quadruple dcl mutants exposed DCL1 as a sophisticated enzyme capable of producing both microRNAs (miRNAs) and siRNAs, unlike the three other DCLs, which only produce siRNAs. Depletion of siRNA-specific DCLs results in unbalanced small RNA levels, indicating a redeployment of DCL/DRB complexes. In particular, DCL2 antagonizes the production of miRNAs and siRNAs by DCL1 in certain circumstances and affects development deleteriously in dcl1 dcl4 and dcl1 dcl3 dcl4 mutant plants, whereas dcl1 dcl2 dcl3 dcl4 quadruple mutant plants are viable. We also show that viral siRNAs are produced by DCL4, and that DCL2 can substitute for DCL4 when this latter activity is reduced or inhibited by viruses, pointing to the competitiveness of DCL2. Given the complexity of the small RNA repertoire in plants, the implication of each DCL, in particular DCL2, in the production of small RNAs that have no known function will constitute one of the next challenges.     

Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs

Arabidopsis encodes four DICER-like (DCL) proteins. DCL1 produces miRNAs, DCL2 produces some virus-derived siRNAs, and DCL3 produces endogenous RDR2-dependent siRNAs, but the role of DCL4 is unknown. We show that DCL4 is the primary processor of endogenous RDR6-dependent trans-acting siRNAs (tasiRNAs). Molecular and phenotypic analyses of all dcl double mutants also revealed partially compensatory functions among DCL proteins. In the absence of DCL4, some RDR6-dependent siRNAs were produced by DCL2 and DCL3, and in the absence of DCL3, some RDR2-dependent siRNAs were produced by DCL2 and DCL4. Consistent with partial redundancies, dcl2 and dcl3 mutants developed normally, whereas dcl4 and dcl3 dcl4 mutants had weak and severe rdr6 phenotypes, respectively, and increased tasiRNA target mRNA accumulation. After three generations, dcl3 dcl4 and dcl2 dcl3 mutants exhibited stochastic developmental phenotypes, some of which were lethal, likely owing to the accumulated loss of heterochromatic siRNA-directed marks. dcl1 dcl3 and dcl1 dcl4, but not dcl1 dcl2 mutants, had phenotypes more severe than dcl1 mutants, consistent with DCL1, DCL3, and DCL4 acting as the primary processors of the three respective classes of endogenous silencing RNAs and DCL2 acting to produce viral-derived siRNAs and as an alternative DCL for endogenous siRNA production.     

Arabidopsis DRB4 protein in antiviral defense against Turnip yellow mosaic virus infection

RNA silencing is an important antiviral mechanism in diverse eukaryotic organisms. In Arabidopsis DICER‐LIKE 4 (DCL4) is the primary antiviral Dicer, required for the production of viral small RNAs from positive‐strand RNA viruses. Here, we showed that DCL4 and its interacting partner dsRNA‐binding protein 4 (DRB4) participate in the antiviral response to Turnip yellow mosaic virus (TYMV), and that both proteins are required for TYMV‐derived small RNA production. In addition, our results indicate that DRB4 has a negative effect on viral coat protein accumulation. Upon infection DRB4 expression was induced and DRB4 protein was recruited from the nucleus to the cytoplasm, where replication and translation of viral RNA occur. DRB4 was associated with viral RNA in vivo and directly interacted in vitro with a TYMV RNA translational enhancer, raising the possibility that DRB4 might repress viral RNA translation. In plants the role of RNA silencing in viral RNA degradation is well established, but its potential function in the regulation of viral protein levels has not yet been explored. We observed that severe infection symptoms are not necessarily correlated with enhanced viral RNA levels, but might be caused by elevated accumulation of viral proteins. Our findings suggest that the control of viral protein as well as RNA levels might be important for mounting an efficient antiviral response.     

A Turnip crinkle virus Isolate Lacking the CP Counter‐Defence Protein Gene Providing Protection Against the Wild‐Type Strain is Associated with Highly Localized RNA Silencing

Cross‐protection has been used successfully and commercially to control a range of virus diseases for which the selection of suitable mild strains of plant viruses is necessary. Turnip crinkle virus (TCV) is highly pathogenic on Arabidopsis plants and its silencing suppressor‐defective mutant, TCVΔCP, can induce highly localized RNA silencing which is differs from that of other protective strains. We found that TCVΔCP provides some protection against wild‐type TCV but lacks complete protection, and the relative locations of the protective virus and challenge virus affect the degree of cross‐protection. However, similar cross‐protection afforded by TCVΔCP is not observed in Nicotiana benthamiana plants. As expected, TCVΔCP pre‐infected Arabidopsis plants fail to protect against infection with the unrelated Cucumber mosaic virus, strain Fhy. It appears that cross‐protection afforded by TCVΔCP requires that the challenge virus be very similar in sequence, which is a characteristic of RNA silencing. In order to investigate whether the protection is associated with the highly localized RNA silencing, mutant plants involved in key silencing pathway genes of RNA silencing machinery, including dcl2, dcl4 and triple dcl2/dcl3/dcl4 mutants were used. The results demonstrate that cross‐protection afforded by TCVΔCP is dependent on host RNA silencing, and both DCL2 and DCL4 play important roles in this process.     

The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis

It has been reported that some double-stranded RNA (dsRNA) binding proteins interact with small RNA biogenesis-related RNase III enzymes. However, their biological significance is poorly understood. Here we examine the relationship between the Arabidopsis microRNA- (miRNA) producing enzyme DCL1 and the dsRNA binding protein HYL1. In the hyl1-2 mutant, the processing steps of miR163 biogenesis were partially impaired; increased accumulation of pri-miR163 and reduced accumulation of short pre-miR163 and mature miR163 as well as misplaced cleavages in the stem structure of pri-miR163 were detected. These misplaced cleavages were similar to those previously observed in the dcl1-9 mutant, in which the second double-stranded RNA binding domain of the protein was disrupted. An immunoprecipitation assay using Agrobacterium-mediated transient expression in Nicotiana benthamiana showed that HYL1 was able to form a complex with wild-type DCL1 protein, but not with the dcl1-9 mutant protein. We also examined miR164b and miR166a biogenesis in hyl1-2 and dcl1-9. Increased accumulation of pri-miRNAs and reduced accumulation of pre-miRNAs and mature miRNAs were detected. Misplaced cleavage on pri-miR164b was observed only in dcl1-9 but not in hyl1-2, whereas not on pri-miR166a in either mutant. These results indicate that HYL1 has a function in assisting efficient and precise cleavage of pri-miRNA through interaction with DCL1.     

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.     

The rice yellow mottle virus P1 protein exhibits dual functions to suppress and activate gene silencing

In plants RNA silencing is a host defense mechanism against viral infection, in which double‐strand RNA is processed into 21–24‐nt short interfering RNA (siRNA). Silencing spreads from cell to cell and systemically through a sequence‐specific signal to limit the propagation of the virus. To counteract this defense mechanism, viruses encode suppressors of silencing. The P1 protein encoded by the rice yellow mottle virus (RYMV) displays suppression activity with variable efficiency, according to the isolates that they originated from. Here, we show that P1 proteins from two RYMV isolates displaying contrasting suppression strength reduced local silencing induced by single‐strand and double‐strand RNA in Nicotiana benthamiana leaves. This suppression was associated with a slight and a severe reduction in 21‐ and 24‐nt siRNA accumulation, respectively. Unexpectedly, cell‐to‐cell movement and systemic propagation of silencing were enhanced in P1‐expressing Nicotiana plants. When transgenically expressed in rice, P1 proteins induced specific deregulation of DCL4‐dependent endogenous siRNA pathways, whereas the other endogenous pathways were not affected. As DCL4‐dependent pathways play a key role in rice development, the expression of P1 viral proteins was associated with the same severe developmental defects in spikelets as in dcl4 mutants. Overall, our results demonstrate that a single viral protein displays multiple effects on both endogenous and exogenous silencing, not only in a suppressive but also in an enhancive manner. This suggests that P1 proteins play a key role in maintaining a subtle equilibrium between defense and counter‐defense mechanisms, to insure efficient virus multiplication and the preservation of host integrity.     

Differential contributions of plant Dicer‐like proteins to antiviral defences against potato virus X in leaves and roots

Members of the plant Dicer‐like (DCL) protein family are the critical components of the RNA‐silencing pathway that mediates innate antiviral defence. The distinct antiviral role of each individual DCL protein has been established with mostly based on observations of aerial parts of plants. Thus, although the roots are closely associated with the life cycle of many plant viruses, little is known about the antiviral activities of DCL proteins in roots. We observed that antiviral silencing strongly inhibits potato virus X (PVX) replication in roots of some susceptible Solanaceae species. Silencing of the DCL4 homolog in Nicotiana benthamiana partially elevated PVX replication levels in roots. In Arabidopsis thaliana, which was originally considered a non‐host plant of PVX, high levels of PVX accumulation in inoculated leaves were achieved by inactivation of DCL4, while in the upper leaves and roots, it required the additional inactivation of DCL2. In transgenic A. thaliana carrying the PVX amplicon with a green fluorescent protein (GFP) gene insertion in the chromosome (AMP243 line), absence of DCL4 enabled high levels of PVX‐GFP accumulation in various aerial organs but not in the roots, suggesting that DCL4 is critical for intracellular antiviral silencing in shoots but not in roots, where it can be functionally compensated by other DCL proteins. Together, the high level of functional redundancies among DCL proteins may contribute to the potent antiviral activities against PVX replication in roots.     

GTSF‐1 is required for formation of a functional RNA‐dependent RNA Polymerase complex in Caenorhabditis elegans

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte‐specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C‐terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the Caenorhabditis elegans Gtsf1 homolog, named it gtsf‐1 and characterized it in the context of the sRNA pathways of C. elegans. We report that GTSF‐1 is not required for Piwi‐mediated gene silencing. Instead, gtsf‐1 mutants show a striking depletion of 26G‐RNAs, a class of endogenous sRNAs, fully phenocopying rrf‐3 mutants. We show, both in vivo and in vitro, that GTSF‐1 interacts with RRF‐3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF‐1 is required for the assembly of a larger RRF‐3 and DCR‐1‐containing complex (ERIC), thereby allowing for 26G‐RNA generation. We propose that GTSF‐1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA‐mediated silencing activities.     

Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E

RNA sequencing studies have identified hundreds of non‐coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high‐throughput analysis of RNA–RNA interactions in bacteria. Here we demonstrate that in vivo sRNA–mRNA duplexes can be recovered using UV‐crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base‐paired sRNA–mRNA duplexes in association with RNase E, allowing proximity‐dependent ligation and sequencing of cognate sRNA–mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA–mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co‐regulated target mRNAs. We identified multiple mRNA targets for the pathotype‐specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli. Numerous sRNA interactions were also identified with non‐coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.     

Oryza sativa dicer-like4 reveals a key role for small interfering RNA silencing in plant development

MicroRNAs and small interfering RNAs (siRNAs) are two classes of small regulatory RNAs derived from different types of precursors and processed by distinct Dicer or Dicer-like (DCL) proteins. During evolution, four Arabidopsis thaliana DCLs and six rice (Oryza sativa) DCLs (Os DCLs) appear to have acquired specialized functions. The Arabidopsis DCLs are well characterized, but those in rice remain largely unstudied. Here, we show that both knockdown and loss of function of rice DCL4, the homolog of Arabidopsis DCL4, lead to vegetative growth abnormalities and severe developmental defects in spikelet identity. These phenotypic alterations appear to be distinct from those observed in Arabidopsis dcl4 mutants, which exhibit accelerated vegetative phase change. The difference in phenotype between rice and Arabidopsis dcl4 mutants suggests that siRNA processing by DCL4 has a broader role in rice development than in Arabidopsis. Biochemical and genetic analyses indicate that Os DCL4 is the major Dicer responsible for the 21-nucleotide siRNAs associated with inverted repeat transgenes and for trans-acting siRNA (ta-siRNA) from the endogenous TRANS-ACTING siRNA3 (TAS3) gene. We show that the biogenesis mechanism of TAS3 ta-siRNA is conserved but that putative direct targets of Os DCL4 appear to be differentially regulated between monocots and dicots. Our results reveal a critical role of Os DCL4-mediated ta-siRNA biogenesis in rice development.     

Deformability in the cleavage site of primary microRNA is not sensed by the double‐stranded RNA binding domains in the microprocessor component DGCR8

The prevalence of double‐stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA‐binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD‐containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8‐Core, consists of its two dsRBDs and a C‐terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N‐terminal dsRBD of DGCR8 in isolation nor the DGCR8‐Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single‐stranded segments flanking the stem. Extending DGCR8‐Core to include an N‐terminal heme‐binding region does not change our conclusions. Thus, our data suggest that although the DGCR8‐Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency. Proteins 2015; 83:1165–1179. © 2015 Wiley Periodicals, Inc.