Abstract

Dicers (Dcrs) are central proteins involved in the biogenesis of small RNAs (sRNAs). Most of the knowledge on Dcr structure, function and evolution comes from studies in animals and plants. Comparatively, less is known in fungi, a genetically and ecologically diverse group with important roles in ecosystems, agriculture, medicine, and biotechnology. While canonical Dcrs contain a well-defined domain architecture, most fungal Dcrs lack one or more identifiable canonical domains, raising questions about how RNA-binding and sRNA-processing is retained. We conducted an extensive survey of fungal Dcrs, analyzing 1592 proteomes across nine phyla. We found a diversity of domain architectures, with some lacking identifiable Piwi, Argonaute and Zwille (PAZ), Helicase, and/or double-stranded RNA-binding (dsRBD) domains. Phylogenetic analyses showed that different Dcr classes are distributed across distinct clades that often align with fungal taxonomic groups. Despite the lack of canonical domain architectures, fungal Dcrs fold into characteristic structures and show PAZ-like folds, displaying an OB-fold core typical of PAZ domains. Molecular simulation analyses further indicate that these divergent Dcrs maintain key RNA-binding surfaces for proper sRNA processing. Our results indicate a remarkable evolutionary plasticity of Dcr in fungi, showing that essential sRNA processing functions can be retained through structural conservation, and highlighting fungi as models to study the modular evolution of the RNAi machinery in eukaryotes. © 2025 The Authors