Biology subjects

Lisitskaya, L.

Publications and source records attributed to Lisitskaya, L..

5 recordsLinked to original sources

bioRxiv2026

Tetramerization and RNA-guided filament assembly control Schlafen-Argonaute antiphage defense

Two deeply conserved protein families, Argonaute (Ago) and Schlafen (SLFN), play defense roles in diverse prokaryotes and eukaryotes, including humans. Here, we identify a monophyletic group of proteins broadly distributed across bacteria and archaea that fuse a SLFN domain with an Ago core and a GHKL-family ATPase. Using SLFN-pAgo from Runella zeae as a model, we show that these proteins protect bacterial cells against bacteriophages by employing the Ago core as a guide-dependent sensor and the SLFN domain as a nuclease effector to induce abortive infection via tRNAs cleavage. Structural and biochemical analyses reveal that ATP binding by the GHKL domain drives tetramerization of SLFN-pAgo, reconstituting the canonical SLFN nuclease architecture found in mammalian proteins. Prior to target detection, non-canonical guide RNA binding induces the formation of long helical filaments, locking the SLFN domains in an inactive configuration. Guide-dependent recognition of complementary target DNA triggers massive structural rearrangements leading to filament disassembly and the induction of SLFN nuclease activity. Together, our findings uncover a new antiphage system that employs reversible guide- and ATP-mediated oligomerization to strictly regulate cooperation between an Ago sensor and a SLFN RNAse effector.

molecular biology↗

bioRxiv2025

Targeting of human mitochondrial DNA with programmable pAgo nuclease

Manipulating the mitochondrial genome remains a major challenge in genetic engineering, largely due to the mitochondrial double-membrane structure. While recent advances have expanded the genetic toolkit for nuclear and cytoplasmic targets, precise editing of mtDNA has remained elusive. Here we report the first successful mitochondrial import of a functional RNA-guided prokaryotic Argonaute protein from the mesophilic bacterium Alteromonas macleodii (AmAgo). By directing AmAgo to the single-stranded D- or R-loop region of mtDNA using synthetic RNA guides, we observed a nearly threefold reduction in mtDNA copy number in human cell lines. This proof of concept study demonstrates that a bacterial Argonaute can remain active within the mitochondrial environment and influence mtDNA levels. These findings lay the groundwork for further development of programmable systems for mitochondrial genome manipulation. HighlightsAmAgo targeted to mitochondria significantly reduces human mtDNA copy number Mitochondrial localization of AmAgo is essential for changes in the mtDNA content AmAgo effects on mtDNA are modulated by exogenous guide RNAs Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/689640v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@ddd069org.highwire.dtl.DTLVardef@9db9a3org.highwire.dtl.DTLVardef@1068b02org.highwire.dtl.DTLVardef@904c87_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Target-induced Argonaute-HNH filaments confer bacterial immunity

Argonaute proteins provide innate immunity in all domains of life through guide-dependent recognition of invader nucleic acids. While eukaryotic Argonautes (eAgos) act on RNA during RNA interference, prokaryotic Argonautes (pAgos) mainly recognize DNA targets. Many eAgos and some pAgos are active nucleases that directly cleave their targets. In contrast, short pAgos lack the nuclease activity and are co-encoded with additional effectors. Diverse effector domains in short pAgo systems include NADases and tentative nucleases, but their mechanisms of activation remain largely unknown. Here, we characterize SPARHA systems (short prokaryotic argonautes, HNH-associated) encoding HNH nuclease effectors. We show that short pAgo activates the HNH effector after RNA-guided DNA recognition. Target recognition induces formation of filaments of SPARHA with double active sites formed at the interfaces of repetitive HNH domains, which results in indiscriminate collateral degradation of DNA and protects bacterial population from invaders. The results show that pAgos and associated effectors act as modular two-component systems that translate recognition of specific DNA into immune response through assembly of supramolecular complexes, deleterious for invaders and potentially useful for biotechnology.

molecular biology↗

bioRxiv2023

DNA-targeting short Argonaute triggers effector nuclease to protect bacteria from invaders

Two prokaryotic defence systems, Argonautes (pAgos) and CRISPR-Cas, detect invader nucleic acids using complementary guides. Upon recognition, the target is cleaved through nuclease activities of pAgo or Cas proteins thus protecting the cell from invasion. However, not all pAgos are active nucleases. Members of a large clade of short pAgos bind nucleic acid guides but lack nuclease activity suggesting a different mechanism of action. Here, we have investigated short pAgo from Novosphingopyxis baekryungensis (NbaAgo). We have shown that NbaAgo forms a heterodimeric complex, SPARDA, with a co-encoded effector nuclease. RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA results in cell death during plasmid transformation or phage infection, thus protecting bacterial population from invaders. The collateral activity of SPARDA allows highly sensitive detection of specific DNA targets. SPARDA expands the list of prokaryotic immune systems that elicit suicidal cell response with a unique range of nuclease activities, creating additional opportunities for biotechnologies.

molecular biology↗

bioRxiv2022

Bacterial Argonaute nucleases reveal different modes of DNA targeting in vitro and in vivo

Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes (eAgos) that were similarly proposed to play a role in cell defense against invaders. However, pAgos are much more diverse than eAgos and very little is known about their functional activity and target specificity in vivo. Here, we describe five pAgo proteins from mesophilic bacteria that act as DNA-guided DNA endonucleases and analyze their ability to target chromosomal and invader DNA. In vitro, the analyzed proteins use small guide DNAs for precise cleavage of single-stranded DNA at a wide range of temperatures. Upon their expression in Escherichia coli, all five pAgos are loaded with small DNAs preferentially produced from plasmid DNA and from chromosomal regions of replication termination. One of the tested pAgos, EmaAgo from Exiguobacterium marinum can induce DNA interference between multicopy sequences resulting in targeted processing of homologous plasmid and chromosomal loci. EmaAgo also protects bacteria from bacteriophage infection and is preferentially loaded with phage guide DNAs suggesting that the ability of pAgos to target multicopy elements may be crucial for their protective function. The wide spectrum of pAgo activities suggests that they may have diverse functions in vivo and paves the way for their use in biotechnology.

molecular biology↗