Periodic Reporting for period 3 - COMPASS (Comprehending Prokaryotic Argonaute Systems)
Reporting period: 2023-12-01 to 2025-05-31
In this ERC project we investigate the role and mechanisms of short prokaryotic Argonaute proteins. Proteins from the Argonaute protein family can be programmed with a small nucleic acid guide to recognize complementary nucleic acid sequences. The role of Argonaute proteins is well described in eukaryotes, where they play an essential role in regulation of gene expression. Prokaryotes (bacteria and archaea) also encode Argonaute proteins (pAgos), but these homologs show a much greater diversity in sequence and domain composition, and pAgo-encoding genes genetically cluster with other proteins in operons, suggesting functional links. It has previously been shown that certain 'stand alone' long pAgos proteins function as immune systems that are programmed to recognize and neutralize invading DNA such an plasmids and viruses. However, the function and mechanisms of truncated 'short' Argonaute proteins was completely unexplored. In this project we aim to uncover roles and mechanisms of various pAgo variants. This will provide a fundamental understanding of how pAgos can confer immunity against invading nucleic acids. Furthermore, given the ability to (re)program pAgos with nucleic acid guides to bind nucleic acids with complementary sequences, their catalytic properties could be repurposed for programmable genome editing and/or nucleic acid sequence detection purposes, thereby expanding the arsenal of programmable molecular tools.
In our first publication, (https://www.wur.nl/en/newsarticle/new-immune-system-discovered-in-bacteria.htm(opens in new window) we describe the role and mechanism of short prokaryotic Argonaute TIR-APAZ (SPARTA) systems. In these systems, a short pAgo genetically associates with a TIR-APAZ protein. Together, these proteins form a SPARTA complex that enables RNA-guided detection of complementary DNA sequences. Upon detection of invading DNA sequences, the TIR domain is catalytically activated, and catalyzes the conversion of NAD+ to ADPR and NAM. In a bacterial host, SPARTA detects the plasmid of invading plasmid DNA and kills its host by depletion of the metabolite NAD+. This prevents spread of the invading plasmid DNA through the bacterial population, and as such SPARTA protects the bacterial population against nucleic acid invaders. As SPARTA can be isolated and reprogrammed with synthetic RNA guides, it can be repurposed for detection of specific nucleic acid sequences.
Furthermore, we have performed detailed bioinformatics analyses to further chart the diversity of short pAgos, and have described that truncation of long pAgos occurred at multiple evolutionary events (https://edepot.wur.nl/582249(opens in new window)). We have reviewed the current insights in the rapidly developing short pAgo field and have pointed out differences between different short pAgo systems and presented major remaining questions in the field.
Finally, we have analyzed similarities and differences between Argonautes encoded in prokaryotes and eukaryotes (https://www.sciencedirect.com/science/article/pii/S1369527423000504?via%3Dihub(opens in new window)). While eukaryotic and prokaryotic Argonautes can confer immunity, and the general mechanism (nucleic acid-guided detection of nucleic acids), the mechanisms of different Argonaute proteins as well as the pathways in which they are involved vary widely.
Furthermore, we have performed detailed bioinformatics analyses to further chart the diversity of short pAgos, and have described that truncation of long pAgos occurred at multiple evolutionary events (https://edepot.wur.nl/582249(opens in new window)). We have reviewed the current insights in the rapidly developing short pAgo field and have pointed out differences between different short pAgo systems and presented major remaining questions in the field.
Finally, we have analyzed similarities and differences between Argonautes encoded in prokaryotes and eukaryotes (https://www.sciencedirect.com/science/article/pii/S1369527423000504?via%3Dihub(opens in new window)). While eukaryotic and prokaryotic Argonautes can confer immunity, and the general mechanism (nucleic acid-guided detection of nucleic acids), the mechanisms of different Argonaute proteins as well as the pathways in which they are involved vary widely.
In the remainder of this project, we aim to further characterize SPARTA systems and the role and mechanisms of other uncharacterized pAgo systems.