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Structural and mechanistic studies of RNA-guided and RNA-targeting antiviral defense pathways

Final Report Summary - ANTIVIRNA (Structural and mechanistic studies of RNA-guided and RNA-targeting antiviral defense pathways)

The conflict between viruses and their host cells occurs in all domains of life. While cells have evolved sophisticated antiviral restriction systems, viruses respond with their own mechanisms to evade detection and targeting by these systems. The aim of the ERC Starting Grant ANTIVIRNA is to study the molecular mechanisms of antiviral pathways in which RNA plays a central role, focusing on (i) bacterial CRISPR-associated nucleases and (ii) eukaryotic RNA processing and modification pathways implicated in innate immunity and viral evasion.

CRISPR-Cas systems, found in many bacteria, are adaptive RNA-guided “immune” systems that target genetic parasites such as viruses. At the core of these systems are protein-RNA complexes that function as sequence-specific DNA-cutting enzymes (DNases). In a subset of CRISPR systems, the protein Cas9 associates with an unusual guide RNA composed of two RNA molecules (crRNA and tracrRNA) and cleaves a double-stranded target DNA target sequence matching the sequence of the guide RNA. The programmability of Cas9 has been exploited in the development of a powerful molecular technology for editing the genomes of cells and organisms. To shed light on the molecular mechanism of Cas9, X-ray crystallography was used to determine atomic structures of the enzyme in its autoinhibited free state, and in a complex with a guide RNA and a target DNA. The structures revealed that the enzyme undergoes conformational changes upon guide RNA binding that enables it to bind the target DNA and uses a base-specific recognition mechanism to bind a short sequence motif in the target DNA (termed the PAM motif), which facilitates hybridization between the guide RNA and the target DNA strand. The structural studies of Cas9 have not only provided critical insights into the molecular mechanism of the enzyme but also catalysed the development of engineered Cas9 variants with improved specificity or non-canonical DNA recognition properties, thereby enabling a new generation of genome editing tools and technologies.

The mammalian innate immune system recognizes molecular signatures of viruses and activates the expression of antiviral effector proteins. In turn, viruses have evolved mechanisms to evade the innate immune system of the host. RNase L is a potent antiviral effector that is allosterically activated by 2’,5’-oligoadenylate, molecular signal synthesized in cells upon detection of viral double-stranded RNA. Several pathogenic viruses evade this restriction mechanism using phosphodiesterase enzymes that break down 2-5A. A crystal structure of one of these enzymes, determined within the framework of the project, provided insights into its catalytic mechanism and evolutionary history. Another subset of viruses such as influenza innate immunity by inhibiting the 3’ end polyadenylation of host mRNAs, a critical process in eukaryotic mRNA biogenesis which depends on the recognition of a specific polyadenylation signal sequence in the precursor mRNA transcript by the cleavage and polyadenylation specificity complex (CPSF). Crystallographic and cryo-EM analysis of the CPSF complex, undertaken in the context of the project, revealed the molecular basis of polyadenylation signa recognition and suggested how viruses might inhibit this process. Finally, methylation of adenine bases (m6A) is an emerging RNA modification with important roles in regulating the processing, translation and stability of cellular and viral RNAs. Crystallographic studies of the core methylation enzyme (METTL3/METTL14 complex) revealed its molecular architecture and mechanism of substrate RNA binding. Together, the structural and mechanistic studies of RNA recognition and processing pathways implicated in innate immunity and antiviral advanced our understanding of their basic molecular mechanisms and provided the foundation for their targeting in cellular and antiviral therapies.