Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis

Abstract

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....

Arabidopsis histone deacetylase 6: a green link to RNA silencing

“…Their biogenesis and function is controlled by a specialized set of RNAi proteins, which are DICER-LIKE 3 (DCL3), the RNA-dependent RNA polymerase RDR2 and the Argonaute protein AGO4 Xie et al, 2004). In addition, siRNA generation and function is dependent on RNA polymerase IV, which is unique to plants (Herr et al, 2005;Kanno et al, 2005;Onodera et al, 2005;Pontier et al, 2005) and a plant-specific SNF2-like protein, DRD1 (Kanno et al, 2004). Pol IV exists as two distinct complexes in Arabidopsis, which are functionally diverse and specified by their unique largest subunits, respectively.…”

“…Pol IV exists as two distinct complexes in Arabidopsis, which are functionally diverse and specified by their unique largest subunits, respectively. PolIVa is required for biogenesis of siRNAs, whereas PolIVb and DRD1 act downstream of siRNAs to mediate de novo DNA methylation by a yet unknown mechanism (Kanno et al, 2005;Pontier et al, 2005). The nuclear-acting siRNAs are processed from their long dsRNA precursors in the nucleolus, where they colocalize with the proteins mentioned above except for PolIVa subunits and DRD1.…”

Short Silencing RNA: The Dark Matter of Genetics?

“…These mutant phenotypes also indicate that sRNA production by pol IVb is dependent on pol IVa but not vice versa (Pontier et al 2005). A plausible mechanism that explains this interdependence is similar to the amplification of tasiRNAs: A primary siRNA produced in a pol IVa pathway would cleave a pol IVb transcript so that it becomes a template for dsRNA production by an RDR.…”

“…These isoforms share a common second-largest subunit, but they have largest subunits with different carboxy-terminal domains. From the siRNA phenotype of RNA silencing mutants, it seems that pol IV provides a template RNA that is converted into dsRNA by an RDR and then into siRNA by a DCL (Herr et al 2005;Pontier et al 2005).…”

Repeat elements and the Arabidopsis DNA methylation landscape

“…In the next step, the DNA MTase DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is the most active Arabidopsis homolog of mammalian de novo DNA MTases Goll and Bestor, 2005), is recruited by the Pol V-AGO4-siRNA complex in a sequence-specific manner to establish methylation at CG, CHG and CHH sites (Cao and Jacobsen, 2002b;Cao et al, 2003). Pol V and AGO4 also contribute to the accumulation of siRNAs at some loci ( Figure 1a), suggesting either a reinforcement loop (Kanno et al, 2005;Pontier et al, 2005;Mosher et al, 2008) or a role for these proteins in siRNA stabilization. Several additional proteins are implicated in de novo DNA methylation, such as the SRA-and SET-domain-containing histone methyltransferases SUPPRESSOR OF VARIEGA-TION 3-9 HOMOLOGUE 2 (SUVH2) and SUVH9 as well as the putative transcription elongation and chromatinremodelling factors SUPPRESSOR OF TY INSERTION 5-LIKE (SPT5L, also known as KTF1) and DEFECTIVE IN RNA-DIRECTED DNA METHYLATION (DRD1), but their precise function is less well understood (Law and Jacobsen, 2010).…”