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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....

A sequence element located immediately upstream of the TATA element, and having the consensus sequence 5-G/C-G/C-G/A-C-G-C-C-3, affects the ability of transcription factor IIB to enter transcription complexes and support transcription initiation. The sequence element is recognized directly by the transcription factor IIB. Recognition involves ␣-helices 4 and 5 of IIB, which comprise a helix-turn-helix DNA-binding motif. These observations establish that transcription initiation involves a fourth core promoter element, the IIB recognition element (BRE), in addition to the TATA element, the initiator element, and the downstream promoter element, and involves a second sequence-specific general transcription factor, IIB, in addition to transcription factor IID. Efficient transcription initiation at a human protein-encoding gene requires assembly on promoter DNA of a multiprotein complex containing RNA polymerase II (Pol II) and six general transcription factors, IIA, IIB, IID, IIE, IIF, and IIH (for review, see Orphanides et al. 1996;Roeder 1996;Nikolov and Burley 1997). Previous work establishes that assembly of the complex involves three core promoter elements: (1) the TATA element, located near position −30, (2) the initiator element, located near position −1, and (3) the downstream promoter element, located near position +30 (Burke and Kadonaga 1996;Orphanides et al. 1996;Roeder 1996). Transcription factor IID is responsible for recognition of at least two of these elements. One subunit of IID, TATA-binding protein (TBP), is responsible for recognition of the TATA element; one or more of the remaining subunits of IID, TBP-associated factors (TAFs), is responsible for recognition of the downstream promoter element. It is not clear which factor is responsible for recognition of the initiator element.The crystallographic structure of a ternary complex of transcription factor IIB core domain (IIBc), TBP core domain (TBPc), and a 16-bp DNA fragment containing the TATA element shows that IIBc interacts with both TBPc and DNA, interacting with the DNA major groove immediately upstream of the TATA element and the DNA minor groove immediately downstream of the TATA element ( Fig. 1; Nikolov et al. 1995). In the crystallographic structure, details of the interaction between IIBc and the DNA major groove upstream of the TATA element are incomplete, since the structure was determined using a DNA fragment containing only three nucleotide pairs upstream of the TATA element (Nikolov et al. 1995). However, DNA-binding and site-specific protein-DNA photocross-linking experiments confirm that interaction between IIB and the DNA major groove upstream of the TATA element occurs and indicate that the interaction is extensive, spanning up to 7-9 nucleotide pairs (Lee and Hahn 1995;Lagrange et al. 1996). The observation that IIB makes extensive interactions with the DNA major groove upstream of the TATA element raises the possibility that the ability of IIB to enter into transcription complexes and, thus, the ability of IIB to support...

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.

ARGONAUTE4 (AGO4) and RNA polymerase IV (Pol IV) are required for DNA methylation guided by 24 nucleotide small interfering RNAs (siRNAs) in Arabidopsis thaliana. Here we show that AGO4 localizes to nucleolus-associated bodies along with the Pol IV subunit NRPD1b; the small nuclear RNA (snRNA) binding protein SmD3; and two markers of Cajal bodies, trimethylguanosine-capped snRNAs and the U2 snRNA binding protein U2B''. AGO4 interacts with the C-terminal domain of NRPD1b, and AGO4 protein stability depends on upstream factors that synthesize siRNAs. AGO4 is also found, along with the DNA methyltransferase DRM2, throughout the nucleus at presumed DNA methylation target sites. Cajal bodies are conserved sites for the maturation of ribonucleoprotein complexes. Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci.