Argonaute protein slicing, retention of RNA fragments is a key role in chemical modification of DNA for gene silence.

Researchers from Indiana University Bloomington discovered previously unknown steps in a gene silencing process that is used to fight viruses and other genome invaders. The team behind the new findings published in Genes & Development.

Researchers from Indiana University Bloomington discovered previously unknown steps in a gene silencing process that is used to fight viruses and other genome invaders. The team of researchers led by Craig Pikaard, Distinguished Professor of Biology and Molecular and Cellular Biochemistry published their findings in Genes & Development. The study shows how ARGONAUTE 4, a member of a significant protein family, binds, cuts, and retains snippets from ribonucleic acids (RNA) molecules that guide chemical inactivation for genes with matching sequences.

Gene silencing mediated through RNA molecules is known by RNA interference (or RNAi). It can occur in many organisms, including animals, plants, insects, and fungi. There are many types of RNAi. However, they all share some common features. All RNAi begin with RNA polymerase proteins interpreting the genetic information in DNA and copying it to RNA. This is known as transcription. Double-stranded RNAs are synthesized in all RNAi pathways. The two strands are paired as a DNA double-helix. These dsRNAs can be cut by Dicer proteins to shorter dsRNAs. The individual strands of these RNAs can vary from 21-35 nucleotides (the individual units RNA polymers), depending on the species. The dsRNAs are then sliced and loaded into an Argonaute protein. Only one strand (the guide strand) is intended to remain stably linked with the AGO protein. The passenger strand is released and degraded. The AGO protein uses the guide string to identify RNA targets to whom the guide strand could pair, leading to gene suppression by different methods. The guide programs AGO are used to inactivate RNAs which encode proteins. They either cut the targetRNA into two fragments, or sequester it away from the protein synthesizing machine. A second scenario is where the guide RNA and target RNA are paired while the target gene is still being synthesized. This allows for the recruitment of proteins that chemically alter the gene being transscribed. These modifications can be performed in a variety of organisms, including plants and humans. They involve adding single-carbon methyl group to the transcribed DNA. This causes changes in gene organization that stop further rounds of transcription.

Wang et. al. Wang et al. studied AGO4's involvement in RNAi pathways in plants called RNA directed DNA methylation. This pathway's dicer enzyme generates both 23- and 24-nucleotide (nt), RNAs. However, prior studies only identified 24 ntRNAs that were associated with AGO4, leaving the function of the 23-ntRNAs unclear. Analyzing plants engineered to produce AGO4 revealed a plant that was unable to cut target RNAs. This line found that AGO4 was associated with both 23 and 24 nucleotide RNAs. This suggests that 23 ntRNAs are normally passenger strands for 24ntRNAs and are then cut, which was confirmed by a testtube simulation. Surprise! The fragments of the sliced 23-nt RNAs weren't released as expected. They were instead retained by AGO4 in both the cell and the testtube. Wang and colleagues continued to investigate this observation and found that target RNA fragments were also retained by AGO4 even after slicing. This suggests that these fragments play an important role in RNA-directed DNAmethylation. The authors confirmed this notion by finding that AGO4's DNA slicing activity is required to achieve high levels at targeted sites throughout the genome. The authors suggest that retained RNA fragments may help to tether AGO4 RNA complexes with the corresponding DNA sequences, increasing the efficiency of DNA methylation.