Investigation of Aspergillus fumigatus small RNA biogenesis uncovers evidence of double-stranded RNA-dependent growth arrest

ABSTRACT Aspergillus fumigatus is a ubiquitous filamentous fungus and dangerous human pathogen that produces a limited pool of small RNAs, consisting in large part of tRNA-derived RNAs (tDRs). Here, we improve our understanding of the small RNAs produced in conidia and mycelium of A. fumigatus using small RNA-sequencing of strains lacking RNA interference (RNAi) machinery and a cutting-edge tDR-sequencing approach. We find little evidence of small RNAs dependent on the canonical RNAi machinery under laboratory growth conditions, but reveal tDRs to be differentially abundant across fungal morphotypes, with specific fragments proving dominant in each assessed condition (e.g., Asp(GTC)-5’tRH in conidia; His(GTG)-5’tRH in mycelium). Consistent with the literature, we observed distinct patterns of tDRs from nuclear- and mitochondria-derived tDRs, which was confirmed for wild-type fungus with tDR-seq. By inducing canonical RNAi with overexpression of an inverted-repeat transgene, we determined A. fumigatus to produce predominantly 20-nt, 5’ uridine-containing small RNAs from the transgene, a population reliant on Argonaute and Dicer-like proteins. Surprisingly, we found that overexpression of this double-stranded RNA (dsRNA) limited growth of both wild-type and RNAi-deficient strains, with a strain lacking the two A. fumigatus RNA-dependent RNA polymerase orthologs particularly vulnerable. Dicer-like- and Argonaute-double knockout strains with few detectable transgene-derived small RNAs were also susceptible to growth inhibition, suggesting excessive dsRNA limits growth in A. fumigatus . Ultimately, we have provided an improved description of small RNA biogenesis in A. fumigatus and uncovered an intriguing link between dsRNA metabolism and filamentous growth. IMPORTANCE The mechanisms of small RNA biogenesis in fungi are diverse and multifactorial. Here, using a panel of RNA interference gene deletion strains and multiple small RNA sequencing approaches, we reveal limited input of the canonical RNAi machinery on the small RNA landscape of the human fungal pathogen, Aspergillus fumigatus under normal growth conditions. We identify specific patterns of tRNA-derived RNAs unique to fungal morphotypes, which may hold promise for future diagnostic efforts. We then probed the mechanism of double-stranded RNA processing by the organism after artificial induction, revealing a potential link between proper double-stranded RNA processing and fungal growth arrest, improving our understanding of the evolution of double-stranded RNA regulation in a ubiquitous mold.