Pontes found the other half of the team located within a special nuclear compartment known as the nucleolus, long known to be the production center for ribosomes. "She got a brilliant signal in the nucleolus, a brilliant dot in the same place for each of the proteins," said Pikaard. Using RNA-FISH, Pontes also found that the siRNAs were in that same dot within the nucleolus.
Pontes and Pikaard were able to deduce the order of events by studying mutations of all eight genes that give rise to the proteins, finding out what happens to the different proteins as the different genes are mutated, one by one. For instance, the researchers found the importance of RNA Polymerase IVa (Pol IVa) by looking at a Pol IVa mutant and noting that the rest of the proteins didn't localize properly. In the RNA-dependent RNA polymerase 2 (RDR2) mutant, Pol IVa is unaffected, but the function of all the other proteins downstream is lost, inferring that it came into the act second. The picture that emerged from this logical approach is that Pol IVa gets things started, churning out RNA that then goes to the nucleolus where it is acted on by RDR2, which turns the single-stranded RNA into double-stranded RNA. The Dicer-like 3 protein, DCL3 then chops the RNA into small interfering RNAs (siRNAs). Along comes ARGONAUTE4 (AGO4), which grabs hold of the siRNAs while also binding to NRPD1b, the largest subunit of an alternative form of RNA Polymerase IV, Pol IVb. The AGO4-siRNA-NRPD1b complex is then thought to leave the nucleolus, acquire the second-largest Pol IV subunit, NRPD2, which serves both Pol IVa and Pol IVb, and then seek out the DNA sequences that match the siRNAs. At these sites, the chromatin remodeler DRD1 presumably bulldozes histones and other proteins out of the way to make the DNA accessible for methylation by the de novo cytosine methyltransferase, DRM2.
Source:Washington University in St. Louis