Functional characterization of Argonaute syndromes variants in Caenorhabditis elegans

Abstract

Precise regulation of gene expression is essential for animal development and physiology. Argonaute (AGO) proteins are central to gene regulatory control as core components of microRNA (miRNA)-mediated post-transcriptional silencing. De novo coding variants in human AGO genes have been identified as causative for rare developmental disorders collectively termed Argonaute syndromes (AS), which encompass a broad spectrum of clinical severity. As the number of distinct AS variants continues to grow, there is a need to rapidly define how individual variants affect the molecular functions of AGO proteins and link these molecular defects to developmental outcomes. To address this need, I leveraged the tractable genetic model organism Caenorhabditis elegans to functionally characterize an expanded set of AS variants in vivo. Building on previous work that modeled four AGO1 AS variants in the C. elegans homolog alg-1, I used CRISPR/Cas9 genome editing to engineer 14 additional alg-1 AS alleles. By integrating genetic and molecular analyses, I examined how the modeled clinical AGO variants perturb miRNA-mediated gene regulation during development. Characterization of this expanded variant set revealed a broad range of functional consequences. While some alg-1 AS alleles caused only mild developmental defects, others produced more severe phenotypes than complete loss of alg-1, exhibiting antimorphic behavior that was sensitive to wild-type alg-1 dosage. Molecular analyses further showed that the modeled AS variants produced allele-specific miRNA abundance and transcriptome profiles that were generally distinct from those of the alg-1 null mutant. Together, these findings indicate that AS variants differentially disrupt AGO function through mechanisms that extend beyond simple loss of activity. This work reinforces the utility of C. elegans as a powerful in vivo platform for rapid functional analysis of clinical AGO variants and gives new insights into how individual variants perturb AGO function. In addition, the strains and approaches presented here provide a foundation for future studies aimed at defining variant-specific molecular mechanisms and identifying genetic pathways and pharmacological interventions that modify the effects of AGO dysfunction. Such rapid molecular characterizations of AS variants can inform potential therapeutic strategies for Argonaute syndromes.