INVESTIGATION OF RASOPATHY VARIANTS AND RAF PROTEIN REGULATION

Abstract

RASopathies are a group of human developmental disorders caused by mutations in genes encoding components of the ERK signaling pathway, including RAF1 and BRAF. Although most RASopathy-associated variants enhance ERK signaling, the underlying mechanisms remain poorly understood. We hypothesize that variants affecting different domains of RAF1 and BRAF may alter signaling through distinct mechanisms, such as changes in Raf protein expression or subcellular localization. To investigate these possibilities, we used Caenorhabditis elegans and CRISPR-Cas9 gene editing to analyze the protein properties of GFP-tagged LIN-45, the only Raf ortholog in C. elegans. We examined the effects of five disease-associated variants that alter conserved regions within the cysteine-rich domain (CRD), conserved region 2 (CR2), kinase domain (KD), or C-terminal domain (CTD). We found that the CRD and CR2 domains influence LIN-45 localization in adult germline. Specifically, mutations in either the CRD or CR2 resulted in enrichment of LIN-45 at the apical membrane, while mutations in the kinase domain or C-terminal domain did not alter localization between the apical membrane and cytoplasm. In addition, we found that mutations in the CRD, kinase domain, and C-terminal domain decrease LIN-45 protein abundance. Moreover, we discovered that mutations in the CRD, CR2, KD, and CTD caused quantitative gain-of-function phenotypes in the C. elegans germline, similar to those observed in a Ras gain-of-function mutant. These findings suggest that, as in human cells, RASopathy-associated variants act as gain-of-function alleles in C. elegans. Finally, the gain-of-function phenotypes displayed by the C. elegans mutants are consistent with increased actin–myosin contractility, suggesting a possible role for Raf in regulating cytoskeletal function. In summary, we found that C. elegans is a useful model for assessing RASopathy variants and suggest that this approach will reveal cellular roles of Raf signaling that are relevant to RASopathy disease symptoms.