Wednesday, January 15, 2025
1/15/2025
PROJECT SUMMARY CRISPRs (Clustered, Regularly, Interspersed, Palindromic Repeats) and CRISPR-associated proteins (Cas) are essential components of adaptive immune systems in bacteria and archaea that provide sequence-specific protection from viruses and other genetic parasites. CRISPR systems are phylogenetically and functionally diverse. Most of the work on CRISPR systems has focused on a subset of systems that function as RNA- guided endonucleases that exclusively bind and cleave double-stranded DNA (dsDNA). In all CRISPR subtypes, this RNA guide is derived from the CRISPR loci transcripts and serves as a genetic memory of past infections. The work in this application focuses on determining the structure and function of a unique type III CRISPR system that includes Cas genes that contain domain fusions bearing strong homology to known toxins (HEPN and RelE) and an antitoxin (MntA). Unlike most CRISPR systems, that target DNA, type III CRISPR systems are unique in their utilization of multi-subunit ribonucleoprotein complexes to bind and destroy RNA that is complementary to the CRISPR-RNA guide. In addition to targeting RNA, type III CRISPR systems encode a specialized surveillance complex subunit called Cas10. Target recognition by type III triggers polymerase-cyclase activity in the palm domain of the Cas10 subunit which amplifies the immune response by selectively converting ≈4,000 ATP molecules into cyclic oligoadenylates that function as immune signaling molecules. Using computational methods, the Wiedenheft lab has recently identified a unique subclass of type III-B CRISPRs distributed across three phyla. These type III-B systems have co-opted elements of toxin- antitoxin systems and the immune cassette includes an additional toxin that I predict is regulated by cyclic oligoadenylate signaling. I hypothesize that these type III-B CRISPR-toxin-antitoxin defense systems rely on CRISPR RNA-guided recognition of invading RNA to activate a multipronged cell death response. In Aim 1, I will use cryo-electron microscopy to determine structures of the type III-B surveillance complexes in various states of activation. These structures will provide critical mechanistic insight that explains how different elements of the immune response are regulated by the effector complex. In Aim2, I will determine the mechanisms by which specialized toxins stall viral infections via a CRISPR-controlled collapse of translation. Collectively, these experiments and the associated training objectives outlined in this application will clarify CRISPR-mediated mechanisms that regulate cell death in response to viral infection.
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