Antiviral signalling by a cyclic nucleotide activated CRISPR protease

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• CRISPR defense systems, including DNA-targeting Cas9 and RNA-targeting type III, are common in prokaryotes.
• Type III CRISPR systems initiate antiviral responses via cyclic oligoadenylate synthesis upon foreign RNA detection.
• Proteins associated with type III systems, including CRISPR-associated Lon protease (CalpL), are predicted to bind cyclic oligoadenylates.

Purpose of the Study:

• To elucidate the structure and function of CRISPR-associated Lon protease (CalpL).
• To understand the mechanism by which CalpL interacts with other proteins in the CRISPR-associated complex.
• To establish the link between CRISPR-mediated foreign nucleic acid recognition and downstream regulatory events.

Main Methods:

• Structural analysis of the CalpL protein.
• Biochemical assays to determine the function of CalpL within a tripartite complex.
• Operon analysis to identify interacting proteins (CalpT and CalpS).

Main Results:

• CalpL forms a stable tripartite complex with CalpT and CalpS.
• Activated CalpL oligomerizes and cleaves CalpT, a MazF homolog.
• Cleavage of CalpT releases the extracytoplasmic function sigma factor CalpS.
• CalpL possesses a SAVED domain that binds cyclic tetra-adenylate (cA4).

Conclusions:

• The study demonstrates a direct link between CRISPR detection of foreign nucleic acids and transcriptional regulation.
• The findings reveal a connection between CRISPR effectors with SAVED domains and cyclic-oligonucleotide-based antiphage signaling systems.
• CalpL's function highlights a novel mechanism for antiviral defense and gene regulation in prokaryotes.