5' UTR mediated post-transcriptional regulation of sexual development in Cryptococcus neoformans by auto-regulatory PUM1
Kaur, Jan Naseer
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Proteins in the PUF (Pumilio and FBF) family of RNA binding proteins bind to 3'UTRs of mRNAs and are associated with regulatory functions including translation, stabilization and localization of transcripts. Investigation of the C. neoformans genome has revealed that it encodes four PUF proteins. PUF proteins are typically characterized by the presence of 8 consecutive PUF repeats; however, variations do occur. The PUF proteins characterized to date have been reported to bind to a consensus sequence encompassing a UGUR tetra nucleotide in their target RNA. Recent phylogenetic studies have demonstrated that RNA binding domain of Puf3, the homolog of C. neoformans Pum1, is conserved and there is significant enrichment of Puf3 binding elements in genes encoding the mitochondrial translation machinery in Saccharomycotina species, which is lost in higher fungi. Our studies indicate that C. neoformans pum1Δ exhibit no detectable mitochondrial phenotype. Phenotypic characterization of a C. neoformans pum1Δ strain revealed a defect in filamentation that led us to hypothesize that Pum1 plays a role in morphogenesis. When equal numbers of opposite mating cells of pum1Δ mutants were co-cultured on V8 agar, we observed that they were defective in filamentation. Using fluorescence microscopy, we showed that mCherry tagged Pum1 specifically localizes to areas of hyphal growth, but is not visible in yeast cells. RNA EMSA demonstrated that Pum1 protein specifically binds to a consensus UGUACAUA cis element present in its own 5'UTR. We demonstrated that Pum1 is auto-regulatory, controlling its own morphotype expression in addition to the downstream process of post-fusion hyphal extension during sexual development. The RNA binding activity of Pum1 was found to be essential for its regulatory function. Deletion of the Pum1 consensus binding element in the 5' UTR of PUM1 leads to hyper-filamentous phenotype, thereby priming the cells for mating, suggesting that PUM1 exerts a repressive role during vegetative growth. The absence of Pum1 severely attenuated the induction of the master filamentation regulator, ZNF2, in a synthetic PGAL7ZNF2 over-expression strain through an indirect mechanism affecting mRNA stability. During hyphal extension, Znf2 drove PUM1 expression from an alternative transcription start site downstream of the Pum1 binding element, relieving Pum1 auto-repression. These data support a model in which Pum1 is repressive for filamentation during vegetative growth through auto-regulation, and is supportive of filamentation in part through indirect stabilization of ZNF2, which in turn drives PUM1 expression during sexual development. Thus, the interplay of transcription factor, Znf2 and the post-transcriptional regulator, Pum1 fine-tunes the downstream mating process in C. neoformans.