Those little tykes, just 24 nucleotides long, are somehow responsible for methylation of DNA sequences that match the sequence of the siRNAs, but not without a lot of help from their friends. The friends in this case are the team of eight known proteins of the RNA-directed DNA methylation pathway.
Using an impressive toolkit of sophisticated techniques, Pikaard and his collaborators not only have described the locations of the eight proteins in the pathway but also have provided the sequence of events that leads to methylation. It is a twisted, and ultimately circular path, but Pikaard and his colleagues are the first researchers to literally see the pathway and thereby provide a clearer understanding of the steps leading to methylation and gene silencing.
The results were published in the July 14, 2006 issue of Cell. The study was funded by the National Institutes of Health, Howard Hughes Medical Institute (HHMI) and Monsanto Company. Pikaard's collaborators include Olga Pontes, the first author of the study, other group members from his Washington University laboratory and the group of Steven E. Jacobsen, Ph.D., an HHMI investigator and professor of biology at the University of California, Los Angeles.
Using mutants, antibodies, and fluorescence microscopy techniques known as RNA fluorescence in situ hybridization (RNA-FISH) and DNA-FISH, Washington University postdoctoral researcher Olga Pontes, Ph.D., was able to unravel where the eight team players are located and in what order events in the RNA-directed DNA methylation pathway transpire. Using antibodies to detect the proteins, together with DNA-FISH to detect the DNA sites that give rise to the siRNAs, Pontes found that half of the team is located with the genes that match the siRNAs.
"The combination of DNA FISH and protein localization allowed us to say which proteins are sitting on the DNA that give rise to th
Source:Washington University in St. Louis