The preferred route for the degradation of silencing target RNAs in transgenic plants depends on pre-established silencing conditions

RNA silencing can be initiated upon dsRNA accumulation and results in homology-dependent degradation of target RNAs mediated by 21–23 nt small interfering RNAs (siRNAs). These small regulatory RNAs can direct RNA degradation via different routes such as the RdRP/Dicer- and the RNA-induced silencing complex (RISC)-catalysed pathways. The relative contribution of both pathways to degradation of target RNAs is not understood. To gain further insight in the process of target selection and degradation, we analysed production of siRNAs characteristic for Dicer-mediated RNA degradation during silencing of mRNAs and chimeric viral RNAs in protoplasts from plants of a transgenic tobacco silencing model line. We show that small RNA accumulation is limited to silencing target regions during steady-state mRNA silencing. For chimeric viral RNAs, siRNA production appears dependent on pre-established cellular silencing conditions. The observed siRNA accumulation profiles imply that silencing of viral target RNAs in pre-silenced protoplasts occurs mainly via a RISC-mediated pathway, guided by (pre-existing) siRNAs derived from cellular mRNAs. In cells that are not silenced at the time of infection, viral RNA degradation seems to involve Dicer action directly on the viral RNAs. This suggests that the silencing mechanism flexibly deploys different components of the RNA degradation machinery in function of the prevailing silencing status.     

The CaMV transactivator/viroplasmin interferes with RDR6-dependent trans-acting and secondary siRNA pathways in Arabidopsis

Several RNA silencing pathways in plants restrict viral infections and are suppressed by distinct viral proteins. Here we show that the endogenous trans-acting (ta)siRNA pathway, which depends on Dicer-like (DCL) 4 and RNA-dependent RNA polymerase (RDR) 6, is suppressed by infection of Arabidopsis with Cauliflower mosaic virus (CaMV). This effect was associated with overaccumulation of unprocessed, RDR6-dependent precursors of tasiRNAs and is due solely to expression of the CaMV transactivator/viroplasmin (TAV) protein. TAV expression also impaired secondary, but not primary, siRNA production from a silenced transgene and increased accumulation of mRNAs normally silenced by the four known tasiRNA families and RDR6-dependent secondary siRNAs. Moreover, TAV expression upregulated DCL4, DRB4 and AGO7 that mediate tasiRNA biogenesis. Our findings suggest that TAV is a general inhibitor of silencing amplification that impairs DCL4-mediated processing of RDR6-dependent double-stranded RNA to siRNAs. The resulting deficiency in tasiRNAs and other RDR6-/DCL4-dependent siRNAs appears to trigger a feedback mechanism that compensates for the inhibitory effects.     

An SNF2 protein associated with nuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis

The silencing phenotype in Arabidopsis thaliana lines with an inverted repeat transgene under the control of a phloem-specific promoter was manifested in regions around veins due to a mobile signal of silencing. Genetic analysis implicates RNA-DEPENDENT RNA POLYMERASE2 (RDR2) and an RNA polymerase IVa subunit gene (NRPD1a) in the signaling mechanism. We also identified an SNF2 domain-containing protein (CLASSY1) that acts together with RDR2 and NRPD1a in the spread of transgene silencing and in the production of endogenous 24-nucleotide short interfering RNAs (siRNAs). Cytochemical analysis indicates that CLASSY1 may act in the nucleus with NRPD1a and RDR2 in the upstream part of RNA silencing pathways that generate a double-stranded RNA substrate for Dicer-like (DCL) nucleases. DCL3 and ARGONAUTE4 act in a downstream part of the pathway, leading to endogenous 24-nucleotide siRNA production, but are not required for intercellular signaling. From genetic analysis, we conclude that another downstream part of the pathway associated with intercellular signaling requires DCL4 and at least one other protein required for 21-nucleotide trans-acting siRNAs. We interpret the effect of polymerase IVa and trans-acting siRNA pathway mutations in terms of a modular property of RNA silencing pathways.     

Novel modes of protein-RNA recognition in the RNAi pathway

Gene silencing mediated by RNA interference (RNAi) depends on short interfering RNAs (siRNAs) and micro RNAs (miRNAs). These RNAs have unique features, namely a defined size of 19-21 base pairs, and characteristic two-nucleotide single-stranded 3' overhangs and 5' monophosphate groups. These molecular features of siRNAs and miRNAs are produced by RNase III enzymes, which are a hallmark of gene silencing induced by double-stranded RNA. Recent structural studies of components of the RNAi pathway, including PAZ, Piwi and RNase III domains, as well as full-length Argonaute and viral p19 proteins, have revealed distinct and novel modes of sequence-independent recognition of the characteristic features of siRNAs and miRNAs in the RNAi pathway.     

植物RNA沉默的分子机制研究进展

RNA沉默（RNA silencing）是真核生物中的一种抵抗外源遗传因子（病毒、转座子或转基因）及调控基凶表达的防御机制。参与植物RNA沉默的酶及蛋白质主要包括6种RNA依赖的RNA聚合酶、4种Dicer-like（DCL）核酸内切酶和10种Argonautes蛋白。植物中4条RNA沉默途径分别由微小RNA（miRNAs）和3种小干扰RNA（siRNAs）介导,包括反式作用siRNAs（ta－siRNAs）、天然反义siRNAs（natsiRNAs）和异染色质siRNAs（hc-siRNAs）。在植物RNA沉默的系统性传播中,由DCL4或DCL2将dsRNAs裁剪为次级SiRNAS,以放大RNA沉默信号和增强沉默效应。     

Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants

RNA polymerase III (Pol III) as well as Pol II (35S) promoters are able to drive hairpin RNA (hpRNA) expression and induce target gene silencing in plants. siRNAs of 21 nt are the predominant species in a 35S Pol II line, whereas 24- and/or 22-nucleotide (nt) siRNAs are produced by a Pol III line. The 35S line accumulated the loop of the hpRNA, in contrast to full-length hpRNA in the Pol III line. These suggest that Pol II and Pol III-transcribed hpRNAs are processed by different pathways. One Pol III transgene produced only 24-nt siRNAs but silenced the target gene efficiently, indicating that the 24-nt siRNAs can direct mRNA degradation; specific cleavage was confirmed by 5' rapid amplification of cDNA ends (RACE). Both Pol II- and Pol III-directed hpRNA transgenes induced cytosine methylation in the target DNA. The extent of methylation is not correlated with the level of 21-nt siRNAs, suggesting that they are not effective inducers of DNA methylation. The promoter of a U6 transgene was significantly methylated, whereas the promoter of the endogenous U6 gene was almost free of cytosine methylation, suggesting that endogenous sequences are more resistant to de novo DNA methylation than are transgene constructs.     

Endogenous,Tissue-Specific Short Interfering RNAs Silence the Chalcone Synthase Gene Family in Glycine max Seed Coats

Two dominant alleles of the I locus in Glycine max silence nine chalcone synthase (CHS) genes to inhibit function of the flavonoid pathway in the seed coat. We describe here the intricacies of this naturally occurring silencing mechanism based on results from small RNA gel blots and high-throughput sequencing of small RNA populations. The two dominant alleles of the I locus encompass a 27-kb region containing two perfectly repeated and inverted clusters of three chalcone synthase genes (CHS1, CHS3, and CHS4). This structure silences the expression of all CHS genes, including CHS7 and CHS8, located on other chromosomes. The CHS short interfering RNAs (siRNAs) sequenced support a mechanism by which RNAs transcribed from the CHS inverted repeat form aberrant double-stranded RNAs that become substrates for dicer-like ribonuclease. The resulting primary siRNAs become guides that target the mRNAs of the nonlinked, highly expressed CHS7 and CHS8 genes, followed by subsequent amplification of CHS7 and CHS8 secondary siRNAs by RNA-dependent RNA polymerase. Most remarkably, this silencing mechanism occurs only in one tissue, the seed coat, as shown by the lack of CHS siRNAs in cotyledons and vegetative tissues. Thus, production of the trigger double-stranded RNA that initiates the process occurs in a specific tissue and represents an example of naturally occurring inhibition of a metabolic pathway by siRNAs in one tissue while allowing expression of the pathway and synthesis of valuable secondary metabolites in all other organs/tissues of the plant.     

A structural perspective of the protein–RNA interactions involved in virus-induced RNA silencing and its suppression

Small RNAs, including small interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi-associated interfering RNAs (piRNAs), are powerful gene expression regulators. This RNA-mediated regulation results in sequence-specific inhibition of gene expression by translational repression and/or mRNA degradation. siRNAs and miRNAs are generated by RNase III enzymes and subsequently loaded into Argonaute protein, a key component of the RNA induced silencing complex (RISC), to form the core of the RNA silencing machinery. RNA silencing acts as an ancient cell defense system against molecular parasites, such as transgenes, viruses and transposons. RNA silencing also plays an important role in the control of development. In plants, RNA silencing serves as a potent antiviral defense system. In response, many viruses have developed strategies to suppress RNA silencing. The striking sequence diversity among viral suppressors suggests that different viral suppressors could target different components of the RNA silencing machinery at different steps in different suppressing modes. Significant progresses have been made in this field for the past 5 years on the basis of structural information derived from RNase III family proteins, Dicer fragments and homologs, Argonaute homologs and viral suppressors. In this paper, we will review the current progress on the understanding of molecular mechanisms of RNA silencing; highlight the structural principles determining the protein–RNA recognition events along the RNA silencing pathways and the suppression mechanisms displayed by viral suppressors.     

Studies of the interaction of the viral suppressor of RNA silencing protein p19 with small RNAs using fluorescence polarization

Tombusviruses use a 19 kDa protein (p19) as a suppressor of the RNA silencing pathway during infection. The p19 protein binds to short-interfering RNA (siRNA) as a dimer and shows a high selectivity for short duplex RNAs over other RNA species. Since p19 can bind to synthetic and RNA silencing generated small RNAs with little sequence dependence and with size selectivity, this protein has utility as a tool for studying RNA silencing pathways in eukaryotes. However, the ability of p19 to serve as a tool for studying RNA silencing pathways may be complicated by the presence of other endogenous small RNAs such as micro-RNAs (miRNAs). To understand the importance of endogenous small RNA components with respect to p19's ability to bind to siRNAs, we examined the interactions of p19 with human miR-122, a 23-nucleotide duplex miRNA containing several mismatched base pairs that is highly abundant in the liver. The binding characteristics were compared with those of an siRNA optimized against the human kinase CSK. The binding studies were performed using fluorescence polarization experiments on duplex oligonucleotides containing Cy3 dye labels at the 5'-end of one of the strands of RNA as well as electrophoretic gel mobility shift assays. Both methods indicate that the synthetic siRNA with no mismatches in base pairing bound with >3-fold selectivity over that of miR-122. Our results suggest that p19 can distinguish between siRNAs and miRNA species, although the difference in binding constants is not so large that interactions with endogenous miRNAs can be totally ignored.     

Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation.