Analysis of short non-coding RNA–DNA-protein interaction networks in Pyrococcus furiosus

There is growing evidence that non-coding (nc) RNAs play an important role in the regulation of gene expression. They are commonly found associated with specific proteins (RNPs). Argonaute (Ago) proteins interact with specific classes of ncRNAs sp called small ncRNAs or small RNAs. They are the catalytic components of the RNA-induced silencing complex (RISC) (Hammond et al. 2000; Peters and Meister 2007; Hutvagner and Simard 2008; Carthew and Sontheimer 2009) and can be found in all domains of life. Ago proteins are the actual mediators of gene silencing effects whereas the small RNA that associates with Ago complexes serves as sequence-specific guide. We are exploring the function of Ago proteins in Archaea. Archaea organisms do not possess any obvious Dicer protein, which is very important for the production of many small RNA species. So the function of the Ago protein in such organisms is totally unclear. Our model organism is the hyperthermophilic archaeon Pyrococcus furiosus for which a genetic knockout system was recently developed. The archaeal Pyrococcus furiosus Ago (Pf-Ago) surprisingly showed higher binding affinity for single-stranded DNA than for ssRNA. There was no interaction detectable for nucleic acids longer than 35nt. We have generated a potent anti-Pf-Ago antibody and currently, we are using immunoprecipitation (IP) followed by cloning and deep sequencing to identify nucleic acid sequences interacting with Pf-Ago. Our future aims are the characterisation of the nuclease activity in vitro and in vivo. Furthermore, we will analyse if the Pf-Ago interacts with other cellular proteins.