Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense

The mechanisms underlying induction and suppression of RNA silencing in the ongoing plant-virus arms race are poorly understood. We show here that virus-derived small RNAs produced by Arabidopsis Dicer-like 4 (DCL4) program an effector complex conferring antiviral immunity. Inhibition of DCL4 by a viral-encoded suppressor revealed the subordinate antiviral activity of DCL2. Accordingly, inactivating both DCL2 and DCL4 was necessary and sufficient to restore systemic infection of a suppressor-deficient virus. The effects of DCL2 were overcome by increasing viral dosage in inoculated leaves, but this could not surmount additional, non-cell autonomous effects of DCL4 specifically preventing viral unloading from the vasculature. These findings define a molecular framework for studying antiviral silencing and defense in plants.     

Widespread role for the flowering-time regulators FCA and FPA in RNA-mediated chromatin silencing

The RRM-domain proteins FCA and FPA have previously been characterized as flowering-time regulators in Arabidopsis. We show that they are required for RNA-mediated chromatin silencing of a range of loci in the genome. At some target loci, FCA and FPA promote asymmetric DNA methylation, whereas at others they function in parallel to DNA methylation. Female gametophytic development and early embryonic development are particularly susceptible to malfunctions in FCA and FPA. We propose that FCA and FPA regulate chromatin silencing of single and low-copy genes and interact in a locus-dependent manner with the canonical small interfering RNA-directed DNA methylation pathway to regulate common targets.     

拟南芥DCL1启动子的分离及其表达特性分析

Dicer酶是类似于RNase Ⅲ的核酸内切酶,能够特异性地将双链RNA剪切成约20 nt的小RNA,在基因沉默途径中起到非常重要的作用。从拟南芥中分离出DCL1基因上游启动子序列2 kb及DCL1基因的5′端1 kb序列,构建了含有该启动子和GUS报告基因的植物表达载体,通过农杆菌介导法转化拟南芥,并对转基因植株进行GUS 组织化学染色及荧光定量分析,结果表明,在DCL1基因启动子的驱动下,报告基因GUS主要在拟南芥的叶片中表达,在幼嫩的叶片及茎尖中表达量也比较高,茎中的表达量较低,在根中只有微量的表达。     

A link between mRNA turnover and RNA interference in Arabidopsis

In RNA interference (RNAi), double-stranded RNA (dsRNA) triggers degradation of homologous messenger RNA. In many organisms, RNA-dependent RNA polymerase (RdRp) is required to initiate or amplify RNAi, but the substrate for dsRNA synthesis in vivo is not known. Here, we show that RdRp-dependent transgene silencing in Arabidopsis was caused by mutation of XRN4, which is a ribonuclease (RNase) implicated in mRNA turnover by means of decapping and 5'-3' exonucleolysis. When both XRN4 and the RdRp were mutated, the plants accumulated decapped transgene mRNA. We propose that mRNAs lacking a cap structure become exposed to RdRp to initiate or maintain RNAi.     

植物中依赖于RNA的RNA聚合酶研究进展

依赖于RNA的RNA聚合酶(RDR)能够以单链RNA为模版合成互补RNA链,产生双链RNA。双链RNA在细胞内被类似RNaseⅢ的酶DCL加工成20～24 nt的小分子干扰RNA(siRNA)。siRNA可以在转录水平或转录后水平抑制靶基因的表达。RDR通过基因沉默途径参与植物的生长发育调节、逆境应答以及表观遗传修饰等许多生物学过程。加深对植物RDR表达模式、生化活性及生物学功能等方面的理解将有利于植物基因沉默的发展和运用。     

miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay

microRNAs (miRNAs) regulate gene expression through translational repression and/or messenger RNA (mRNA) deadenylation and decay. Because translation, deadenylation, and decay are closely linked processes, it is important to establish their ordering and thus to define the molecular mechanism of silencing. We have investigated the kinetics of these events in miRNA-mediated gene silencing by using a Drosophila S2 cell-based controllable expression system and show that mRNAs with both natural and engineered 3' untranslated regions with miRNA target sites are first subject to translational inhibition, followed by effects on deadenylation and decay. We next used a natural translational elongation stall to show that miRNA-mediated silencing inhibits translation at an early step, potentially translation initiation.     

Quantitative modulation of polycomb silencing underlies natural variation in vernalization

Arabidopsis thaliana accessions have adapted to growth in a wide range of climates. Variation in flowering and alignment of vernalization response with winter length are central to this adaptation. Vernalization involves the epigenetic silencing of the floral repressor FLC via a conserved Polycomb (PRC2) mechanism involving trimethylation of Lys(27) on histone H3 (H3K27me3). We found that variation for response to winter length maps to cis polymorphism within FLC. A rare combination of four polymorphisms localized around the nucleation region of a PHD-Polycomb complex determines a need for longer cold. Chromatin immunoprecipitation experiments indicate that these polymorphisms influence the accumulation of H3K27me3 in Arabidopsis accession Lov-1, both at the nucleation site and over the gene body. Quantitative modulation of chromatin silencing through cis variation may be a general mechanism contributing to evolutionary change.