Reconstitution of human RNA interference in budding yeast

Abstract

Although RNA-mediated interference (RNAi) is a widely conserved process among eukaryotes, including many fungi, it is absent from the budding yeast Saccharomyces cerevisiae. Three human proteins, Ago2, Dicer and TRBP, are sufficient for reconstituting the RISC complex in vitro. To examine whether the introduction of human RNAi genes can reconstitute RNAi in S. cerevisiae, genes encoding these three human proteins were introduced into S. cerevisiae. We observed both siRNA and siRNA- and RISC-dependent silencing of the target gene GFP. Thus, human Ago2, Dicer and TRBP can functionally reconstitute human RNAi in S. cerevisiae, in vivo, enabling the study and use of the human RNAi pathway in a facile genetic model organism.

The Multiple Functions of TRBP, at the Hub of Cell Responses to Viruses, Stress, and Cancer

“…While few in vivo studies have been carried out, results indicate that the presence of TRBP is crucial for RNAi activity. Reconstitution in the yeast Saccharomyces cerevisiae showed that Dicer, TRBP, and Ago2 are necessary and sufficient to recapitulate functional human RNAi (137). Cells derived from tarbp2 Ϫ/Ϫ mice exhibit virtually no RNAi activity, but this activity is restored by exogenous TRBP (30).…”

RNA Interference in Protozoan Parasites: Achievements and Challenges

“…This would inevitably energize any functional genetics/genomics studies of such species. Although for protozoan parasites this is still in the realm of hypothesis, there is a precedent for a successful conversion to RNAi proficiency in Saccharomyces cerevisiae studies (19,63). This was achieved through the use of two different sources of RNAi machinery genes.…”

“…In the first approach, introduction of only two genes (Dicer and Argonaute from a related budding yeast species, S. castellii) was sufficient for the successful downregulation of both a reporter transcript targeted by siRNAs (even in yeast species, long hairpin dsRNA produces siRNAs more efficiently than sense and antisense RNA pairs synthesized from opposing promoters) and endogenous retrotransposons (19). In the second approach, the genes encoding human Dicer, AGO2, and TRBP (TAR RNA-Binding Protein) were capable of reconstituting RNAi for use against reporter green fluorescent protein (GFP) mRNA (63).…”

Roles of mRNA Fate Modulators Dhh1 and Pat1 in TNRC6-dependent Gene Silencing Recapitulated in Yeast

“…Notably, the yeast Pumilio-like protein Puf5/ Mpt5 binds to the CCR4-NOT complex to silence and deadenylate specific mRNAs (44,45), suggesting that the CCR4-NOT complex is involved in sequence-specific post-transcriptional regulation independent of the emergence of miRNAs. Previously, the Bartel and Roth laboratories (46,47) showed that gene silencing by siRNA could be reconstituted in S. cerevisiae by expressing either Saccharomyces castellii Ago1 and Dicer1 or human Ago2, Dicer, and TRBP.…”