Identification and Functional Characterisation of WYLContaining Proteins in prokaryotic immunity.

Abstract

The bacterial CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats) system operates as an adaptive immune system, equipping bacteria with the ability to defend against invading mobile genetic elements. This dynamic interplay between hosts and pathogens has led to significant divergence within CRISPR systems. Beyond the core CRISPR effector complex, bacteria have acquired CRISPR accessory proteins that serve as intermediaries in both innate and adaptive immunity, offering protection against highly virulent intruders. Among the less-studied accessory proteins are WYL domain-containing proteins. The WYL domain displays structural similarities with the SM-fold observed in eukaryotic systems, and are co-occurring with CRISPR effectors in a defence island, although the interplay between CRISPR proteins and WYL-containing proteins in response to phage attack remain elusive. In the current project, a genomic and metagenomic survey was performed on a 30TB of assemblies, with a specific focus on the WYL-containing proteins in the vicinity of CRISPR effectors. This survey identified novel WYL-fusion proteins that were not reported previously. Among these identified proteins a new undescribed clade of large protein from Ruminoclostridium, namely WYLD co-occur specifically with type VI-D CRISPR effectors, targeting RNA is sought to play an important role in prokaryote immunity. WYLD is characterised by two hypothetical HTH domains situated at the N-terminal region, followed by two RNAinteracting regions containing WYL domains, displaying the typical SM fold architecture. Phage assays revealed that WLD alone or in combination with Cas13d restrict phage infections by inducing bacterial dormancy. Mechanistic characterisation demonstrated that WYLD exhibits a nanomolar affinity for GUrich RNA substrate and forms a stable complex with RNA and DNA. This stable RNA-WYLD complex formation and specificity for GU-rich substrate is mediated by key residues in the WYL domain. Additionally, both Cas13d and WYLD exhibit RNA cleavage activity in a stoichiometry dependant manner. In summary, this thesis extends our understanding of the role of WYL-containing proteins associated in CRISPR adaptive immunity and their protective roles against phage attack and shed new lights on the role of WYL-containing proteins in RNA binding and cleavage. Show more