Active Genetics & Molecular Biology Cells, Biochemistry & Physiology

Structure function analysis of the RNAi machinery in Cryptococcus deneoformans

Funder Wellcome Charity-funded
Recipient organisation University of Edinburgh
Funding Not disclosed
Period Oct 2025 — Sept 2028

In plain English

AI plain-English summary

A family of fungi called *Cryptococcus* kills hundreds of thousands of people each year, and a key piece of their biology—a gene-silencing system called RNAi—may control how dangerous they become. RNAi normally helps cells shut down rogue genetic elements called transposons. But some highly infectious *Cryptococcus* strains have lost their RNAi machinery entirely, hinting that losing this system might make the fungi more harmful. No one knows exactly how RNAi works in these fungi, or what else it does beyond controlling transposons. This project aims to find out. The researcher will map the proteins that make up the RNAi machinery in *Cryptococcus*, focusing on the RISC complex that does the actual silencing. Early computer modelling and protein-capture experiments have already flagged several unusual features in key proteins—including an extra tail and a region that may help the machinery grab other proteins. Next steps involve rebuilding the RNAi complexes from scratch in the lab and using cryo-electron microscopy to see their 3D structure. This is fundamental science. There is no immediate medical application. But understanding how RNAi works—or fails—in *Cryptococcus* could eventually explain why some strains turn deadly, and point toward new targets for antifungal drugs.

View original technical description
Cryptococcus is a genus of fungi, containing several species of opportunistic pathogens responsible for a large number of deaths. RNAi is a conserved regulatory mechanism mediated by Argonaute proteins, with a role in silencing transposable elements in Cryptococcus. A highly infectious Cryptococcus strain lacking an RNAi mechanism has suggested a link between infectivity and RNAi, However beyond a role in transposon regulation, the mechanisms and wider functions of RNAi in Cryptococcus remain poorly understood. I aim to characterise the proteins involved in Cryptococcus RNAi, with a particular focus on those involved in the makeup of the essential complexes, including the RISC: a complex responsible for the mechanism of silencing. Initial alignments and modelling of the Argonaute proteins in AlphaFold3 have highlighted some features of interest including an N-terminal extension, and an RG rich insert implicated in interactions with Tudor domain containing proteins in other organisms. Additionally, Argonaute immunoprecipitation followed by mass spectrometry has identified several RNAi associated proteins with undetermined roles in Cryptococcus. Next steps will involve investigating the assembly principles of the RNAi machinery through biochemical reconstitution and structural characterisation (using cryoEM and Cross-Linking Mass Spectrometry), and mechanisms of RNAi mediated silencing using structure guided separation-of-function mutants in cells.

View the original record at the funder ↗

Researchers

Finn Ritchie (EPMC Awardee)

Related Research

Grants with similar aims, by meaning.

Investigating the Argonaute-associated RNAi regulation in the opportunistic fungi Cryptococcus
Mechanisms and roles of RNA interference in Cryptococcus neoformans
Mechanisms and functions of RNA interference in Cryptococcus genome regulation
Structural and Functional Characterisation of the MIWI2 Silencing Complex
The CMR complex for prokaryotic RNA silencing

Original classification

PhD Studentship (Basic)

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