Recent genetic and biochemical studies have revealed the existence in plants of a fourth RNA polymerase, RNAPIV, which mediates siRNA accumulation and DNA methylation-dependent silencing of endogenous repeated sequences. Here, we show that Arabidopsis expresses, in fact, two evolutionarily related forms of RNAPIV, hereafter referred to as RNAPIVa and RNAPIVb. These two forms contain the same second-largest subunit (NRPD2), but differ at least by their largest subunit, termed NRPD1a and NRPD1b. Unlike NRPD1a, NRPD1b possesses a reiterated CTD, a feature that also characterizes the largest subunit of RNAPII. Our data indicate that RNAPIVb is the most abundant form of RNAPIV in Arabidopsis. Selective disruption of either form of RNAPIV indicates that RNAPIVa-dependent siRNA accumulation is not sufficient per se to drive robust silencing at endogenous loci and that high levels of DNA methylation and silencing depend on siRNA that are accumulated through a pathway involving the concerted action of both RNAPIV forms. Taken together, our results imply the existence of a novel two-step mechanism in siRNA synthesis at highly methylated loci, with RNAPIVb being an essential component of a self-reinforcing loop coupling de novo DNA methylation to siRNA production. A major evolutionary distinction separating prokaryotes from eukaryotes is the passage from a unique multisubunit DNA-dependent RNA polymerase enzyme (RNAP) to three complexes (Roeder and Rutter 1969), each responsible for the transcription of a subclass of nuclear DNA sequences (Sentenac 1985). RNA polymerase I (RNAPI) transcribes the repeated genes encoding the large ribosomal RNAs, which represent up to four-fifths of total RNA. RNA polymerase II (RNAPII) transcribes all of the cell protein-coding messenger RNAs (mRNAs) as well as some small nuclear RNAs (snRNAs). RNA polymerase III (RNAPIII) is dedicated to the transcription of a collection of genes whose main common feature is that they encode structural or catalytic RNAs (tRNAs, 5S RNA, snRNA) that are components of protein synthesis, splicing, and tRNA processing apparatuses. It is believed that this triplication event provided the eukaryotic cell with a greater flexibility toward energy-consuming cellular functions such as ribosome synthesis, as well as with more sophisticated means for the regulation of gene expression.Prokaryotic and eukaryotic RNA polymerases are multisubunit enzymes that are evolutionarily related to each other through their largest and second-largest subunits (Ebright 2000;Cramer 2002). The largest subunit (≈160 kDa: Ј; A; RPA1; RPB1; RPC1) contains eight regions conserved in order and sequence (A to H), while the second-largest subunit (≈150 kDa: ; B; RPA2; RPB2; RPC2), contains nine such regions (A to I) (Allison et al. 1985;Sweetser et al. 1987). Although the role of these conserved domains is not yet fully understood, the structure determination of RNAPII suggests that they cooperate in the formation of a single fold cleft containing the active site of the enzyme (Cramer et al....
MAP kinases ͉ Ca ϩ2 ͉ cADP ribose ͉ cGMP ͉ disease resistance
NPR1 is a critical component of the salicylic acid (SA)-mediated signal transduction pathway leading to the induction of defense genes, such as the pathogenesis-related (PR)-1 gene, and enhanced disease resistance. Using a yeast two-hybrid screen, we identified several NPR1-interacting proteins (NIPs). Two of these NIPs are members of the TGA/OBF family of basic leucine zipper (bZIP) transcription factors; this family has been implicated in the activation of SA-responsive genes, including PR-1. Six TGA family members were tested and shown to differentially interact with NPR1: TGA2 and TGA3 showed strong affinity for NPR1; TGA5 and TGA6 exhibited weaker affinity; and TGA1 and TGA4 displayed little or no detectable interaction with NPR1, respectively. Interestingly, the amino-termini of these factors were found to decrease their stability in yeast and differentially affect their apparent affinity toward NPR1. The interacting regions on NPR1 and the TGA factors were also defined. Each of four point mutations in NPR1 that disrupt SA signaling in Arabidopsis completely blocked interaction of NPR1 with TGA2 and TGA3. TGA2 and TGA3 were also found to bind the SA-responsive element of the Arabidopsis PR-1 promoter. These results directly link NPR1 to SA-induced PR-1 expression through members of the TGA family of transcription factors.
In plants and invertebrates, viral-derived siRNAs processed by the RNaseIII Dicer guide Argonaute (AGO) proteins as part of antiviral RNA-induced silencing complexes (RISC). As a counterdefense, viruses produce suppressor proteins (VSRs) that inhibit the host silencing machinery, but their mechanisms of action and cellular targets remain largely unknown. Here, we show that the Turnip crinckle virus (TCV) capsid, the P38 protein, acts as a homodimer, or multiples thereof, to mimic host-encoded glycine/tryptophane (GW)-containing proteins normally required for RISC assembly/function in diverse organisms. The P38 GW residues bind directly and specifically to Arabidopsis AGO1, which, in addition to its role in endogenous microRNA-mediated silencing, is identified as a major effector of TCV-derived siRNAs. Point mutations in the P38 GW residues are sufficient to abolish TCV virulence, which is restored in Arabidopsis ago1 hypomorphic mutants, uncovering both physical and genetic interactions between the two proteins. We further show how AGO1 quenching by P38 profoundly impacts the cellular availability of the four Arabidopsis Dicers, uncovering an AGO1-dependent, homeostatic network that functionally connects these factors together. The likely widespread occurrence and expected consequences of GW protein mimicry on host silencing pathways are discussed in the context of innate and adaptive immunity in plants and metazoans.[Keywords: Argonaute; GW motif; TCV; viral suppressor] Supplemental material is available at http://www.genesdev.org.
Two forms of RNA Polymerase IV (PolIVa/PolIVb) have been implicated in RNA-directed DNA methylation (RdDM) in Arabidopsis. Prevailing models imply a distinct function for PolIVb by association of Argonaute4 (AGO4) with the C-terminal domain (CTD) of its largest subunit NRPD1b. Here we show that the extended CTD of NRPD1b-type proteins exhibits conserved Argonaute-binding capacity through a WG/GW-rich region that functionally distinguishes Pol IVb from Pol IVa, and that is essential for RdDM. Site-specific mutagenesis and domain-swapping experiments between AtNRPD1b and the human protein GW182 demonstrated that reiterated WG/GW motifs form evolutionarily and functionally conserved Argonaute-binding platforms in RNA interference (RNAi)-related components.
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