Characterizing the role of Puf3 in sexual development of Cryptococcus neoformans

Abstract

Mating in the human fungal pathogen Cryptococcus neoformans is one mechanism by which spores, the likely infectious propagules are produced. The multistep sexual development process involves mate recognition followed by plasmogamy, filamentation, karyogamy and meiosis ultimately resulting in the basidiospore production. The Puf (Pumilio and FBF) family of RNA binding proteins is known to bind to 3′UTRs of mRNAs and is associated with regulatory functions including translation, stabilization and localization of transcripts. Scanning of 3′UTRs of all the annotated genes of C. neoformans revealed Puf3 binding consensus sequence in the transcripts involved in pheromone signaling cascade. This led us to hypothesize that Puf3 regulates sexual differentiation of C. neoformans. To test this hypothesis we performed bilateral mating assays for wild type and puf3Δ mutants, lacking the Pumilio domain RNA binding protein Puf3. When equal number of opposite mating cells of puf3Δ mutants was cocultured on V8 agar, they were defective in filamentation as compared to the wild type cross. To determine the ability of puf3Δ mutants to produce pheromones and recognize opposite mating partner, northern blot analysis was done. The RNA obtained from mating colonies of puf3Δ mutants bilateral cross was probed for mating pheromone MFα in comparison to the wild type cross (H99α × KN99a ) and the induction of MFα was found to be normal in puf3Δ mutants mating. Our result suggests that mate recognition does occur when puf3Δ mutants are crossed. Future studies will assess the ability of puf3Δ mutants to fuse and undergo post fusion hyphal extension. Verifying the Puf3 binding to mating pheromone signaling transcripts and analyzing the transcriptional level and stability of these transcripts will lead us to define the role of Puf3 in C. neoformans.