Mechanisms and roles of RNA interference in Cryptococcus neoformans

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RNA interference is a fundamental mechanism of genome regulation mediated by small RNAs and conserved across eukaryotes. It can operate in various ways including transcriptional and post-transcriptional regulation, and plays important roles in controlling gene expression and genome stability. Recent studies suggest that RNAi can also play significant roles in host-pathogen interactions. Much of our understanding of RNAi-mediated transcriptional regulation has come from studies in fission yeast, Schizosaccharomyces pombe. This yeast has a relatively simple RNAi pathway with a limited number of target loci - these features have made S. pombe a good model for molecular analysis of some core aspects of RNAi, but also limit its utility for understanding the full diversity of form and function in RNAi pathways. To address this we have begun to investigate RNAi pathways in another yeast, Cryptococcus neoformans. This basidiomycete is an opportunistic human pathogen (responsible for ~1 million cases of meningitis per year in immunocompromised individuals), and is evolutionarily distant from more commonly studied ascomycetes such as S. pombe. Interestingly, C. neoformans appears to have a more complex RNAi pathway, with two of each of the core components, Dicer and Argonaute, each operating on distinct targets according to our preliminary analyses.The aim of this project is to further investigate RNAi function in Cryptococcus. We will characterise RNAi-related protein complexes, and determine the gene expression programs that are regulated by RNAi, and the mechanisms of regulation (e.g. transcriptional versus post-transcriptional control). We will also look at whether RNAi is involved in changes in gene expression associated with Cryptococcus pathogenicity, and look for any evidence of cross-kingdom RNAi, i.e. Cryptococcus siRNAs being transferred to, and functioning in, host cells, or vice versa. The project will provide training in a broad range of molecular biology techniques including a number of -omics approaches such as small RNA sequencing, CRAC (protein-RNA crosslinking; to identify RNAi target transcripts), proteomics (to identify interacting proteins), and ChIP-seq (to assess whether silencing is associated with changes to chromatin). Communication and presentation skills will be developed through regular participation in lab meetings, journal clubs and seminars.