Recovery of dicer-like 1-late flowering phenotype by miR172 expressed by the noncanonical DCL4-dependent biogenesis pathway

MicroRNAs (miRNAs) act as down-regulators of gene expression, and play a dominant role in eukaryote development. In Arabidopsis thaliana, DICER-LIKE 1 (DCL1) is the main processor in miRNA biogenesis, and dcl1 mutants show various developmental defects at the early stage of embryogenesis or at gamete formation. However, miRNAs responsible for the respective developmental stages of the dcl1 defects have not been identified. Here, we developed a DCL1-independent miRNA expression system using the unique DCL4-dependent miRNA, miR839. By replacing the mature sequence in the miR839 precursor sequence with that of miR172, one of the most widely conserved miRNAs in angiosperms, we succeeded in expressing miR172 from a chimeric miR839 precursor in dcl1-7 plants and observed the repression of miR172 target gene expression. In parallel, the DCL4-dependent miR172 expression rescued the late flowering phenotype of dcl1-7 by acceleration of flowering. We established the DCL1-independent miRNA expression system, and revealed that the reduction of miR172 expression is responsible for the dcl1-7 late flowering phenotype.     

Ectopic DICER-LIKE1 expression in P1/HC-Pro Arabidopsis rescues phenotypic anomalies but not defects in microRNA and silencing pathways

Expression of the viral silencing suppressor P1/HC-Pro in plants causes severe developmental anomalies accompanied by defects in both short interfering RNA (siRNA) and microRNA (miRNA) pathways. P1/HC-Pro transgenic lines fail to accumulate the siRNAs that mediate RNA silencing and are impaired in both miRNA processing and function, accumulating abnormally high levels of miRNA/miRNA* processing intermediates as well as miRNA target messages. Both miRNA and RNA silencing pathways require participation of DICER-LIKE (DCL) ribonuclease III-like enzymes. Here, we investigate the effects of overexpressing DCL1, one of four Dicers in Arabidopsis thaliana, on P1/HC-Pro-induced defects in development and small RNA metabolism. Expression of a DCL1 cDNA transgene (35S:DCL1) produced a mild gain-of-function phenotype and largely rescued dcl1 mutant phenotypes. The 35S:DCL1 plants were competent for virus-induced RNA silencing but were impaired in transgene-induced RNA silencing and in the accumulation of some miRNAs. Ectopic DCL1 largely alleviated developmental anomalies in P1/HC-Pro plants but did not correct the P1/HC-Pro-associated defects in small RNA pathways. The ability of P1/HC-Pro plants to suppress RNA silencing and the levels of miRNAs, miRNA*s, and miRNA target messages in these plants were essentially unaffected by ectopic DCL1. These data suggest that P1/HC-Pro defects in development do not result from general impairments in small RNA pathways and raise the possibility that DCL1 participates in processes in addition to miRNA biogenesis.     

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.     

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.     

The Phaseolus vulgaris miR159a precursor encodes a second differentially expressed microRNA

Plant microRNAs originate from a stem-loop structured single-stranded RNA precursor. Each stem-loop is processed to generate a mature microRNA that is recruited to an ARGONAUTE-containing multi-protein complex to direct silencing of its target mRNA. Here we report that the conserved plant miR159a precursor produces a second 21-nt long RNA with the properties of a microRNA. Its presence in different plant species is supported by its conservation in the stem-loop position and expression as determined by northern blot analysis. We show that successive processing by DCL1 produces this novel microRNA from the same precursor as miR159a. In contrast to the low levels observed in other plant models for the equivalent of miR159.2, in P. vulgaris, the accumulation of miR159.2 is easily detectable and when compared to miR159a, their expression patterns are distinct in different organs and growth conditions. Further evidence of the functionality of miR159.2 comes from its association with silencing complexes as demonstrated by co-immunoprecipitation experiments using an AGO1-specific antibody and processing of an artificial GFP reporter construct containing a complementary target sequence. These results indicate that the second small RNA corresponds to a microRNA, at least partially independent of miR159 activity, and that in plants a miRNA precursor may encode multiple regulatory small RNAs.     

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.     

Controlled RISC loading efficiency of miR168 defined by miRNA duplex structure adjusts ARGONAUTE1 homeostasis

Micro RNAs (miRNAs) are processed from precursor RNA molecules with precisely defined secondary stem-loop structures. ARGONAUTE1 (AGO1) is the main executor component of miRNA pathway and its expression is controlled via the auto-regulatory feedback loop activity of miR168 in plants. Previously we have shown that AGO1 loading of miR168 is strongly restricted leading to abundant cytoplasmic accumulation of AGO-unbound miR168. Here, we report, that intrinsic RNA secondary structure of MIR168a precursor not only defines the processing of miR168, but also precisely adjusts AGO1 loading efficiency determining the biologically active subset of miR168 pool. Our results show, that modification of miRNA duplex structure of MIR168a precursor fragment or expression from artificial precursors can alter the finely adjusted loading efficiency of miR168. In dcl1-9 mutant where, except for miR168, production of most miRNAs is severely reduced this mechanism ensures the elimination of unloaded AGO1 proteins via enhanced AGO1 loading of miR168. Based on this data, we propose a new competitive loading mechanism model for miR168 action: the miR168 surplus functions as a molecular buffer for controlled AGO1 loading continuously adjusting the amount of AGO1 protein in accordance with the changing size of the cellular miRNA pool.     

Mutations in the antiviral RNAi defense pathway modify Brome mosaic virus RNA recombinant profiles

RNA interference (RNAi) mechanism targets viral RNA for degradation. To test whether RNAi gene products contributed to viral RNA recombination, a series of Arabidopsis thaliana RNAi-defective mutants were infected with Brome mosaic virus (BMV) RNAs that have been engineered to support crossovers within the RNA3 segment. Single-cross RNA3-RNA1, RNA3-RNA2, and RNA3-RNA3 recombinants accumulated in both the wild-type (wt) and all knock-out lines at comparable frequencies. However, a reduced accumulation of novel 3' mosaic RNA3 recombinants was observed in ago1, dcl2, dcl4, and rdr6 lines but not in wt Col-0 or the dcl3 line. A BMV replicase mutant accumulated a low level of RNA3-RNA1 single-cross recombinants in Col-0 plants while, in a dcl2 dcl4 double mutant, the formation of both RNA3-RNA1 and mosaic recombinants was at a low level. A control infection in the cpr5-2 mutant, a more susceptible BMV Arabidopsis host, generated similar-to-Col-0 profiles of both single-cross and mosaic recombinants, indicating that recombinant profiles were, to some extent, independent of a viral replication rate. Also, the relative growth experiments revealed similar selection pressure for recombinants among the host lines. Thus, the altered recombinant RNA profiles have originated at the level of recombinant formation rather than because of altered selection. In conclusion, the viral replicase and the host RNAi gene products contribute in distinct ways to BMV RNA recombination. Our studies reveal that the antiviral RNAi mechanisms are utilized by plant RNA viruses to increase their variability, reminiscent of phenomena previously demonstrated in fungi.