Exogenous and endogenous sRNAs derived from plant Dicer-like 4 in the RNAi-depleted yeast, Saccharomyces cerevisiae

Exogenous and endogenous dsRNAs perceived by plant Dicer-like 4 protein in the RNAi-depleted cellular context, Saccharomyces cerevisiae (P. Leonetti, A. Consiglio, D. Arendt, R.P. Golbik, S.Gago-Zachert, L. Rubino, T. Gursinsky, S.E. Behrens, V. Pantaleo).In plants, RNase III Dicer-like proteins (DCLs) act as sensors of dsRNAs and process them into short 21- to 24-nucleotide (nt) (s)RNAs. Plant DCL4 is involved in the biogenesis of either functional endogenous or exogenous (i.e. viral) short interfering (si)RNAs, thus playing crucial antiviral roles. In this study we expressed plant DCL4 in Saccharomyces cerevisiae, an RNAi-depleted organism. The enzyme was shown to be functional in processing dsRNA-like molecules in vitro and in vivo into 21- and 22-nt sRNAs. Conversely, DCL4 did not efficiently process a replicase-assisted viral replicon in vivo, providing evidence that viral RNAs are not accessible to DCL4 in membranes associated in active replication. Worthy of note, in yeast cells expressing DCL4, 21- and 22-nt sRNAs are associated with endogenous loci, thus highlighting and expanding the sense/antisense atlas of S. cerevisiae. The results described herein show that S. cerevisiae can provide insights into the functionality of plant dicers and extend the S. cerevisiae tool to new biotechnological applications.