ABSTRACT The evolutionarily conserved Pumilio domain-containing RNA binding proteins (RBPs) are involved in many steps of post-transcriptional gene regulation, including RNA stability, polyadenylation, deadenylation, and translation. Most RBPs are post-translationally modified by methylation of arginine/glycine-rich domains (RG-box) though the consequences of these modifications are not well known. We determined the arginine methylation and phosphorylation landscape of the Pumilio domain containing RBP Puf4 from the basidiomycete fungus Cryptococcus neoformans . We found that methyl-deficient Puf4 mutants do largely complement critical PUF4 deletion phenotypes, such as resistances to endoplasmic reticulum stress, antifungals, and thermotolerance, but were unable to complement the melanization defect of the puf4 △ mutant. Analysis of the Puf4 interactome revealed temperature-dependent and -independent protein-protein interactions, some of which were altered by the methyl-deficient mutations. Overall, we show that the RNA-binding protein Puf4 exhibits extensive post-translational modifications, particularly arginine methylation that impacts function and protein-protein interactions. IMPORTANCE Post-transcriptional processes play a crucial role in modulating responses to various stressors, including temperature and antifungals. To ensure adaptation and survival, stressors must be accurately sensed, and responses tightly regulated. Post-translational modifications serve as a crucial regulatory layer that orchestrates nuanced stress responses. We highlight extensive methylation and phosphorylation of the RNA-binding protein Puf4 and explore the regulatory roles of select methylated arginines. We also found that the interactome network of Puf4 varies with temperature. Cryptococcus neoformans, a basidiomycete fungus that is also an important human pathogen, is an ideal system to illustrate the evolution of gene regulatory processes and how these kingdom or clade-specific regulatory modules contribute to the pathogenic potential of fungi.
Kalem et al. (Wed,) studied this question.