The molecular network of archaeal argonautes

Ribonucleic acid (RNA) is an important building block of life, as it is formed upon transcription of a gene and further translated into a protein. Furthermore, some viruses carry a RNA genome. In eukaryotic cells, e.g. humans and plants, sophisticated molecular mechanisms are in place that specifically downregulate the number of RNA molecules in the cell. This can lead to the elimination of foreign RNA viruses but also to the regulation of the protein expression of certain host genes. The latter ensures that proteins are expressed to certain levels at specific times, leading to a balanced cell cycle. One of these regulatory mechanisms is the RNA interference pathway (RNAi), which operates via the key enzyme Argonaute (Ago). Agos are among the few enzymes that work in concert with small nucleic acids to exert their specific mechanism: Agos bind to short pieces of RNA, called "guides", that base-pair with a complementary RNA molecule in the cell. The specific base-pairing allows the Ago enzyme to easily identify and bind to a specific target RNA molecule in the cell. While active Agos function like molecular scissors and cut the bound RNA, some Ago variants are inactive for cleavage. These inactive Agos remain bound to the RNA molecule, thereby inhibiting protein synthesis. C ollectively, Agos regulate a plethora of different RNA molecules in the eukaryotic cell even in us humans - which is essential for our proper development and sometimes even survival. Hence, the discovery of the Ago-centered RNAi pathway in eukaryotes is considered a scientific milestone and was awarded the Nobel Prize. Even if Argonaute enzymes have become eukaryotic flagships, they are also found in the simplest and oldest cells on our planet, namely the bacteria and the archaea. Archaea are considered to be the ancestors of the first eukaryotic cell, which means that A gos may have originated in these cells long before the emergence of the first eukaryotes and were later inherited by those. This close relationship raises the question of whether these Ago ancestors also regulate gene expression and virus defense in the archaeal cell, or whether they perform other functions (in addition). Biochemical studies show that archaeal Agos also use small nucleic acids to recognize target molecules. However, unlike in eukaryotes, some archaeal Agos can recognize not only RNA but also DNA using RNA or DNA guides. Apart from the biochemical characterizations, however, the biological function of these ancestral Agos remains largely unexplored. This work aims to characterize the molecular mechanisms and biological roles of selected active and inactive archaeal Agos. By combining in vivo and in vitro studies, a comprehensive picture of the roles of archaeal Agos will be established which will reveal functional differences and similarities between active and inactive archaeal and eukaryotic Agos, which might provide insights into their evolution.

RNA is a key molecule of life: it is made when a gene is transcribed and then translated into a protein. In eukaryotes such as humans and plants, a pathway called RNA interference helps keep RNA levels under control. At the heart of this pathway are Argonaute proteins, which bind short "guide" RNAs that, together with other proteins, base-pair with matching RNAs and cut or block them. This dosage control ensures that proteins are produced at the right levels and times, supporting healthy cells and helping to fight RNA viruses. Similar Argonaute proteins are also found in simpler, single-celled organisms, such as bacteria and archaea. Archaea are considered to be closely related to the ancestors of the first eukaryotic cell, so archaeal Argonautes may represent ancestral forms from which eukaryotic Argonautes evolved. However, how Argonautes function in archaeal cells, and whether they work together with partner proteins like their eukaryotic counterparts, has remained largely unknown. In my Schrödinger project, I set out to uncover the function of archaeal Argonautes and how they are connected to other components in the cell. Using biochemical experiments and studies in living cells, we show that archaeal Argonautes are embedded in a network of proteins and nucleic acids rather than acting in isolation. The detailed characterisation of their interaction partners is ongoing and will be reported in future publications. Building on these findings, colleagues from my Schrödinger host lab (Wageningen University & Research, The Netherlands) and I developed and patented a technology that uses one of the characterized archaeal Argonaute enzymes to "clean up" complex DNA samples. When sequencing a mixed DNA sample, some DNA fragments are much more common than others. These abundant fragments "hide" rarer sequences that may be biologically or medically important. In our approach, we use the archaeal Argonaute to deplete the overrepresented DNA, which improves the detection of rare DNA. This can make it possible to find and decipher the genetic code of a rare microorganism in a complex environmental sample or to detect a rare mutation in the DNA of an organism. In collaboration with the Schleper group (University of Vienna), I also investigated another archaeal system that targets RNA, a type III CRISPR-Cas system. Like other CRISPR-Cas systems, it was previously viewed mainly as a virus defense system. However, our results, currently available as a preprint, suggest that in our model archaeon this system can also degrade and regulate host RNA, reminiscent of eukaryotic Argonautes. This shows that an archaeal immune system can directly control the cell's own gene expression. Together, the findings of my Schrödinger project provide important new insight into archaeal Argonautes and other immune systems, their links to gene regulation, and their potential for applications.