Abstract
Golgi reassembly and stacking protein (GRASP) is required for polysaccharide secretion and virulence in Cryptococcus neoformans. In fungal species, extracellular vesicles (EVs) participate in the export of polysaccharides, proteins and RNA. In the present work, we investigated if EV-mediated RNA export is functionally connected with GRASP in C. neoformans using a graspΔ mutant. Since GRASP-mediated unconventional secretion involves autophagosome formation in yeast, we included the atg7Δ mutant with defective autophagic mechanisms in our analysis. All fungal strains exported EVs but deletion of GRASP or ATG7 profoundly affected vesicular dimensions. The mRNA content of the graspΔ EVs differed substantially from that of the other two strains. The transcripts associated to the endoplasmic reticulum were highly abundant transcripts in graspΔ EVs. Among non-coding RNAs (ncRNAs), tRNA fragments were the most abundant in both mutant EVs but graspΔ EVs alone concentrated 22 exclusive sequences. In general, our results showed that the EV RNA content from atg7Δ and WT were more related than the RNA content of graspΔ, suggesting that GRASP, but not the autophagy regulator Atg7, is involved in the EV export of RNA. This is a previously unknown function for a key regulator of unconventional secretion in eukaryotic cells.
Highlights
Extracellular vesicle (EV) formation and release constitute a ubiquitous export mechanism of proteins, DNA and RNA [1,2]
The C. neoformans strains used in this study included the parental isolate H99 and the mutant strains atg7∆ and grasp∆, which were generated in previous studies by our group [20,28]
We first asked whether the lack of either Golgi reassembly and stacking protein (GRASP) or ATG7 expression would affect the EVs composition
Summary
Extracellular vesicle (EV) formation and release constitute a ubiquitous export mechanism of proteins, DNA and RNA [1,2]. EVs participate in the transport of macromolecules across the cell wall [4,5,6]. Fungal EVs transport a variety of macromolecules including proteins, lipids, glycans, pigments and, as more recently described, RNA [4,6,7,8,9]. It has been hypothesized that EV biogenesis in eukaryotes is a complex process that is regulated at multiple levels [10,11]. GRASP (Golgi reassembly and stacking protein) is a secretion regulator originally characterized in human cells as part of the Golgi cisternae stacking and ribbon formation [12,13]. GRASP is required for protein delivery to the plasma membrane or to the extracellular space by an unconventional pathway that involves autophagosome-like structures [14]
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