Biology subjects

Tamulaitis, G.

Publications and source records attributed to Tamulaitis, G..

3 recordsLinked to original sources

bioRxiv2026

Csx15 is a broad specificity ring nuclease bridging CRISPR-Cas and CBASS cyclic nucleotide signaling systems

Prokaryotic antiviral type III CRISPR-Cas and CBASS systems use cyclic nucleotides as second messengers to activate diverse effector proteins. Whereas CBASS signaling typically triggers abortive infection, type III CRISPR-Cas signaling is tightly regulated through controlled signal production and degradation. Here, we define the molecular mechanism of the CRISPR-associated Csx15 protein family and demonstrate that Csx15 functions as a broad specificity ring nuclease capable of degrading cyclic nucleotide signals from both CRISPR-Cas and CBASS pathways. Structural and biochemical analyses reveal that Csx15 adopts diverse oligomeric states and undergoes ligand-induced conformational transitions that promote filament assembly required for catalytic activity, analogous to filament-forming SAVED-domain effectors. Finally, we provide evidence that Csx15 is a close relative of SAVED domains suggesting that Csx15-like proteins may have also contributed to the evolution of CBASS cyclic nucleotide signaling.

biochemistry↗

bioRxiv2024

Filament formation activates protease and ring nuclease activities of CRISPR SAVED-Lon

To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cAn) signaling to activate various auxiliary effectors, including the CRISPR-associated SAVED-Lon protease CalpL, which forms a tripartite effector system together with an anti-{sigma} factor, CalpT, and an ECF-like {sigma} factor, CalpS. Here we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA4 binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA4 in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity.

biochemistry↗

bioRxiv2017

Targeted RNA knockdown by crRNA guided Csm in zebrafish

RNA interference (RNAi) is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a \"proof of principle\" demonstration that CRISPR endoribonucleases can be used for programmable mRNA transcript degradation. Using zebrafish as the animal model and Csm(crRNA) complexes as the CRISPR endoribonucleases, we have targeted a transgenic EGFP transcript expressed from a variety of promoters. A drastic decrease of fluorescence was achieved in germ cells of the vasa:EGFP line. Weaker effects were also seen in fish lines that express EGFP zygotically. Knockdown was statistically significant in cmcl2:EGFP and fli1:EGFP zebrafish lines at 1 day post fertilization (dpf), but reduced to background levels at 2 dpf. The nkx2.5:EGFP fish line was least susceptible to Csm mediated EGFP knockdown. We conclude that at the present stage, Csm mediated knockdown is already efficient for maternal transcripts, and may compare favorably with morpholinos for such targets in zebrafish.

genetics↗