TheCryptococcusextracellular vesicle transcriptome

Abstract

Extracellular vesicles (EVs) are now recognized as key players in the biology of numerous organisms, including pathogenic fungi. However, studying EVs in these organisms remains challenging. The recent implementation of new protocols to purify EVs in the pathogenic yeastCryptococcus neoformanshas resulted in a more detailed description of their structure and protein composition. Although a few publications describing RNA molecules associated with EVs have already been published, we reasoned that these new protocols would be beneficial for gaining a deeper understanding of the EV transcriptome. We thus purified EVs and confirmed that some RNAs were associated with these EV extracts. Iodixanol gradient analyses also revealed that these RNAs co-sedimented with EVs. We then sequenced these RNAs in parallel with RNAs extracted from the very cells producing these EVs using different types of sequencing libraries. Our data confirm the presence of siRNAs and tRFs associated with EVs, some of which are enriched. We also identified some snoRNAs, which inCryptococcus are mostly borne by coding gene or lncRNA introns.

Protocol for separation of fungal extracellular vesicles using ultracentrifugation from solid medium cultures

“…Recent studies in C. neoformans using Iodixanol gradient revealed that RNAs are co-sedimented with EVs, especially in fractions 6,7 and 8. 10 For C. deuterogattii , the highest particle number was also detected in fraction 7, along with the highest concentration of the major cryptococcal polysaccharide, glucuronoxylomannan. 4 It is worth noting that this approach does have drawbacks, notably the increased time consumption and reduced yield that should be considered case by case.…”