RNA polymerases IV and V (Pol IV and Pol V) are plant-specific polymerases that are required for the biogenesis or function of small interfering RNAs (siRNAs) in the siRNA-directed DNA methylation pathway. This pathway silences repeated genomic sequences, including transposable elements and pericentromeric repeats. We have shown that RNA polymerase IV and/or V function is required to maintain the normal organization of the chromatin within the nucleus, such that mutation of their shared second-largest subunit (NRPD2/NRPE2) causes heterochromatin dispersal. In the figure at left, generated by Dr. Olga Pontes, this dispersal of heterochromatin is apparent in several ways. One is by simple DAPI staining of the nuclear DNA which reveals the most highly condensed heterochromatic DNA as the brightests signals. The heterochromatin in wild-type nuclei normally coalesces into ten or fewer chromocenters that include the centromere repeats and other repeated sequences, such as 5S rRNA gene repeats. Simultaneous localization of 5S rRNA gene loci and centromere repeats using DNA fluorescence in situ hybridization (DNA-FISH) shows that these repeats colocalize with each other and with the DAPI-stained chromocenters in wild-type nuclei. However, in a null mutant for NRPD2/NRPE2, the second-largest subunit of both Pol IV and Pol V, chromocenters are disrupted and 5S genes and centromere repeats no longer colocalize precisely.

Pol IV and Pol V have distinct largest subunits. Although we don't know precisely what either form of pol IV does at a molecular level, we do know that Pol IV acts early in the pathway leading to 24 nt siRNA biogenesis and colocalizes with DNA loci that give rise to 24 nt siRNAs. This can be seen in the figure at right in which the two largest subunits of Pol IV, NRPD1 and NRPD2 have been immunolocalized (green signals) using specific antibodies and the NORs (45S rRNA gene clusters) or 5S gene loci have been localized using DNA-FISH (red signals). There are abundant 24 nt siRNAs that match the 45S and 5S rRNA gene loci. The presence of Pol IV directly at these loci, coupled with our evidence that other components of the pathway leading to the production of 24nt siRNAs are mislocalized when Pol IV is mutated, suggests that Pol IV acts at one of the earliest steps in the pathway.

Other proteins in the pathway leading to the production of 24 nt siRNAs include RNA-DEPENDENT RNA POLYMERASE2 (RDR2), DICER-LIKE3 (DCL3) and ARGONAUTE4. The latter three proteins are present throughout the nucleus but colocalize with one another, as well as with the largest subunit of Pol V (NRPE1), within a distinct site in the nucleolus that includes molecular markers of Cajal bodies. The colocalization of RDR2, DCL3, AGO4 and NRPE1 (previously known as NRPD1b, and labeled this way in the figure) in the Cajal-body-like structure, as determined by pairwise analysis of the proteins (red and green signals), gives rise to yellow dot signals in the nucleolus in the figure at left. In other studies, we showed that siRNAs also co-localize with RDR2, DCL2, AGO4 and NRPE1 within the Cajal-body-like structure, leading us to propose that this site is the processing center for 24nt siRNAs.

Our model for the order of events in the siRNA-directed DNA methylation pathway begins with Pol IV, which physically associates with loci that give rise to abundant 24nt siRNAs. We imagine that Pol IV transcripts are used as templates by RDR2 to generate double-stranded RNAs that are then diced by DCL3. Resulting siRNAs associate with AGO4 and direct the modification of corresponding DNA loci in a Pol V-dependent manner. Because Pol IV is present at loci that are distant from the nucleolus, but RDR2, DCL3 and AGO4 colocalize in the nucleolus, we deduce that RNAs must traffic from the sites where the siRNA precursors originate to the nucleolus. Following siRNA biogenesis and assembly of RISC complexes (RNA induced silencing complexes) in the nucleolus, we further deduce that the complexes must traffic back to the loci that are homologous to the siRNAs to mediate the DNA methylation of these loci. We found recently that Pol V functions by generating transcripts at the target loci, with the assistance of the putative chromatin remodeller, DRD1. Our working model is that siRNAs bound to AGO4 may associate with Pol V transcripts and in this way guide the chromatin modifying machinery to the target loci.

At present we don't have biochemical information concerning the templates or enzymatic products of Pol IV or Pol V but a consideration of structural information and sequence information suggests that Pol IV might have distinct properties as compared to Pol I, II or III. For instance, in the image below, blue spheres denote amino acids that are invariant among the largest subunits of Arabidopsis RNA polymerases I, II and III and yeast (S. cerevisiae) RNA pol II but are different in the largest subunit of Pol IV. In this image, the non-consensus amino acids of pol IV are mapped onto the structure of the largest subunit of a yeast pol II elongation complex, determined by Westover, Bushnell and Kornberg (PDB:1R9T). The DNA template strand (gold), the (incomplete) non-template strand (purple), and the nascent RNA (red) are shown as surface renderings. The active site is located at the base of the RNA-DNA duplex. The view is from the side where the second-largest subunit (not shown) forms the wall of the template channel nearest the viewer. Note that a large number of non-consensus amino acids (blue spheres) line the channel where the template feeds into the active site, which may be consistent with the novel functions of Pol IV compared to Pol II.

By purifying Pol IV and Pol V, determining their complete subunit structures, and working to determine their template requirements (e.g. DNA vs. RNA) in vitro, we hope to understand the functions of Pol IV and Pol V in molecular detail.

For additional details, see the following papers and review articles:

Wierzbicki, Andrzej, Jeremy Haag and Craig S. Pikaard (2008). Noncoding transcription by RNA Polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135:635-648. PDF

Pikaard, Craig S., Jeremy R. Haag, Thomas Ream and Andrzej Wierzbicki (2008). Roles of RNA polymerase IV in gene silencing. Trends Plant Sci. 13:390-397.PDF

Pikaard, Craig S. (2006). Cell biology of the Arabidopsis nuclear siRNA pathway for RNA-directed chromatin modification. Cold Spring Harb Symp Quant Biol. 2006;71:473-80. Pubmed; PDF

Pontes, Olga, Carey Fei Li, Pedro Costa Nunes, Jeremy Haag, Thomas Ream, Alexa Vitins, Steven E. Jacobsen and Craig S. Pikaard (2006). The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126: 79-92. Pubmed PDF

Li, Carey F., Olga Pontes, Mahmoud El-Shami, Ian R. Henderson, Yana V. Bernatavichute, Simon W.-L. Chan, Thierry Lagrange, Craig S. Pikaard, and Steven E. Jacobsen (2006). An ARGONAUTE-containing nuclear processing center co-localized with Cajal bodies in Arabidopsis thaliana. Cell 126:93-106. Pubmed PDF

Onodera, Yasuyuki, Jeremy Haag, Thomas Ream, Pedro Costa Nunes, Olga Pontes and Craig S. Pikaard (2005). Plant Nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120:613-622 Pubmed PDF