M. circinelloides causes the human disease mucormycosis, and the ability to switch between yeast and hyphal growth is key to pathogenesis. By interacting with the prolyl isomerase Fkbp12, the antifungal drug FK506 inhibits hyphal growth via deactivation of the fungal protein phosphatase calcineurin. Calo et al. found that resistance to FK506 readily evolves, often as a result of stable mutations in the drug targets: the FKBA gene (which encodes Fkbp12) or the genes that encode subunits of calcineurin. However, several resistant isolates lacked mutations in these genes and frequently reverted to a sensitive phenotype following removal of the drug. As these mutants also lacked the FKBA mRNA and the Fkbp12 protein, the authors reasoned that resistance might involve RNAi-mediated knockdown of Fkbp12. Consistent with this hypothesis, small RNAs (sRNAs) that were antisense to FKBA were highly expressed in the resistant epimutants but were absent in sensitive revertants, which suggested that RNAi-mediated reversible gene silencing was responsible for resistance to FK506.
So, what is the source of the antisense sRNAs? As they showed perfect complementarity to the mature FKBA sense mRNA (which contains exons only) and frequently spanned exon–exon boundaries, this suggested that they were generated by an RNA-dependent RNA polymerase (RdRp) that uses the mature FKBA sense mRNA as a template for synthesis of the antisense RNA. By screening for epimutations in M. circinelloides mutants that lacked components of the RNAi pathway, the authors found that Argonaute, the two Dicer proteins Dcl1 and Dcl2, and RdRp2 were required to silence the expression of FKBA. However, the trigger for RNAi induction in these epimutants is currently unknown, and a specific stress condition that activates this pathway following FK506 exposure was not identified.
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