In this project we aim to characterize the function of prokaryotic Argonautes. Not only do we investigate the physiological role of these interesting proteins, but additionally we explore their molecular mechanism of action.
Role of Argonaute in Eukaryotes - RNA interference
Apart from the classical non-coding RNA molecules that directly participate in protein synthesis (mRNA, rRNA, tRNA), many additional types of non-coding RNAs have been discovered in eukaryotic organisms, such as microRNA (miRNA) and small interfering RNA (siRNA) (Jinek and Doudna 2009, Carthew and Sontheimer 2009). In Eukaryotes, these small RNAs have been demonstrated to be involved in controlling many cellular processes via different mechanisms such as gene regulation and antiviral defence. Small interfering RNA guides are derived from double-stranded RNAs through cleavage by a nuclease called Dicer. Subsequently, Dicer loads the Argonaute protein with the short dsRNA fragment, resulting in formation of the RNA-induced silencing complex (RISC). After transfer to Argonaute (Ago), the short RNA duplex is unwound and the passenger strand is released. The guide is responsible for base-pairing with a target mRNA. The target is generally degraded via Argonaute its slicing activity, resulting in target degradation. RNA-guided RNA interference is a typically eukaryotic process.
Role of Argonaute in prokaryotes - a novel defense system
12% of all prokaryotes possess a homolog of the above mentioned eukaryotic Argonaute (termed pAgo). Interestingly, no homologs of other RNA-interference proteins are encoded in prokaryotes. To gain insight in the role of pAgo, we performed a bioinformatics analysis with a focus on Argonaute protein sequences, and on the genomic neighborhoods of the respective genes in prokaryotes (Makarova & Van der Oost et al. 2009). pAgo encoding genes frequently cluster with genes encoding proteins involved in the defense against phages and/or plasmids. On the basis of this and the role of Ago in eukaryotes, we have proposed that pAgos are key components of a novel class of defense system.
Indeed, we and other have recently shown that prokaryotic Argonautes play a role in defense against invading nucleic acids (Olovnikov et al., 2013, Swarts et al., 2014). While the catalytically inactive Argonaute from Rhodobacter speroides was demonstrated to perform RNA-guided DNA interference (Olovnikov et al., 2013), thereby lowering plasmid contents in the cell, Thermus thermophilus Argonaute (TtAgo) is capable of cleaving plasmid DNA using DNA guides (Swarts et al., 2014). It preferentially acquires DNA guides targeting plasmid DNA via a poorly understood mechanism, and it is able to lower plasmid transformation efficiencies and intracellular plasmid concentrations (Swarts et al., 2014). Additionally, the molecular cleavage mechanism of TtAgo has been elucidated from different crystal structures, revealing a large part of the TtAgo pathway of DNA-guided DNA interference (Sheng et al., 2014).
However, certain aspects of pAgos remain elusive (e.g. how they evolved from DNA interference in prokaryotes to RNA interference in Eukaryotes, how non-catalytically active Argonautes function, etc). Our aim is to unravel these aspects.
Looking for a BSc or MSc project, which includes various molecular techniques and work with bacteria and bacteriophages? Do not hesitate to contact: John van der Oost.
- Carthew, R.W., Sontheimer, E.J. (2009) Origins and mechanisms of miRNAs and si RNAs. Cell 136, 642-655.
- Jinek, M. and Doudna, J.A. (2009) A three-dimensional view of the molecular machinery of RNA interference. Nature 457, 405-412.
- Makarova, K.S., Wolf, Y.I., Van der Oost, J., Koonin, E.V. (2009) Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements. Biology Direct 4, 29.
- Olovnikov, I., Chan, K., Sachidanandam, R., Newman, D. K., and Aravin, A.A. (2013) Bacterial Argonaute samples the transcriptome to identify foreign DNA. Mol. Cell 51, 594–605.
- Sheng, G., Zhao, H., Wang, J., Rao, Y., Tian, W., Swarts, D.C., Van der Oost, J., Patel, D.J., and Wang, Y. (2013) Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage. PNAS, 111, 652–657 (2013).
- Swarts, D.C., Jore, M.M., Westra, E.W., Zhu, Y., Janssen, J.H., Snijders, A.P., Wang, Y., Patel, D.J., Berenguer, J., Brouns, S.J.J., and Van der Oost, J. (2014) DNA-guided DNA interference by a prokaryotic Argonaute. Nature, 2014.