In an attempt to reconstitute the human RNAi system in budding yeast, three human RNAi genes were introduced into S. cerevisiae, together with the appropriate silencing and reporter constructs. Human Ago2, Dicer and TRBP cDNAs were individually cloned and placed under the control of the inducible GAL1 promoter using the Gateway recombination cloning system. For use in silencing, antisense GFP was cloned under the GAL1 promoter; antisense Ade2 was used as a negative control (A). A GFP-expressing reporter strain was created by integrating the GFP(S65T)-KanMX6 module into the endogenous TDH3 locus (B). The GFP-expressing reporter strain was sequentially transformed with plasmids bearing each of the three human RNAi gene constructs, and the expression of each gene under galactose-induction conditions was confirmed by RT–PCR (A). Expression of RISC genes was not observed when the yeast strains were not induced in galactose. This Ago2/Dicer/TRBP (ADT) strain was transformed with plasmids bearing the antisense GFP silencing constructs, followed by northern blot detection of GFP siRNA (B). Introduction of human Ago2, Dicer and TRBP was sufficient to generate GFP siRNA in S. cerevisiae. Strains expressing all possible pairwise combinations of two RISC components (Ago2/TRBP, Ago2/Dicer and Dicer/TRBP) were also constructed. The GFP siRNA biogenesis was observed in the strain lacking Ago2 and the strain lacking TRBP, but not in the strain lacking Dicer, indicating that Dicer is necessary for siRNA biogenesis and that Ago2 and TRBP are not (B). Flow cytometric analysis was next performed to assess whether the introduced human RNAi genes are capable of silencing GFP gene expression. When the ADT strain expressing antisense-GFP silencing construct was induced with galactose, a significant decrease in the GFP fluorescence intensity was observed, indicating that human RNAi system was successfully reconstituted in S. cerevisiae (). Silencing effects were not observed either under the uninduced condition or when an antisense-ADE2 construct was used in place of the antisense-GFP construct. Strains expressing all possible pairwise combinations of two RISC components (Ago2/TRBP, Ago2/Dicer and Dicer/TRBP) did not exhibit silencing effects (). Taken together, our results show that the three human genes are necessary and sufficient for reconstitution of the human RNAi system in S. cerevisiae.
Figure 1. A schematic diagram showing the silencing antisense constructs and the GFP reporter strain. (A) GFP or Ade2 were each separately cloned under control of GAL1 promoter in the antisense orientation to generate silencing antisense constructs. (B) GFP reporter (more ...)
Figure 2. Expression of human RNAi genes and generation of siRNA in yeast. (A) Expression of human Ago2, Dicer and TRBP genes was assessed by RT–PCR in S. cerevisiae strains expressing either no human RNAi genes (WT) or in an isogenic strain carrying plasmid-borne (more ...)
Figure 3. Gene silencing by human RNAi in yeast. GFP gene silencing was determined by flow cytometric analysis. Histograms show GFP fluorescence in the indicated S. cerevisiae strains (WT or ADT) expressing the indicated silencing constructs (α-ADE2 or (more ...)
Comparison of silencing effects of different combination of RISC components
In this study, we used antisense GFP as a silencing construct. Although hairpin sequences can also be used for this purpose, the GFP hairpin RNA transcribed under the GAL1
promoter may lack a structure that is required for Dicer-mediated processing (16–18
). Although RNA polymerase III promoters are often used for the transcription of shRNAs in mammalian cells (19–21
), the yeast U6 RNA promoter is ill-defined (22
). Antisense GFP RNA should hybridize with the endogenous GFP transcript, and thus generate substrate double-stranded RNA for Dicer-mediated cleavage. Indeed, GFP siRNA was detected by northern blot () as was target gene silencing (). The silencing effect of antisense GFP alone (without the RNAi genes) was negligible (, bottom).
Recently, Drinnenberg et al
) reported the reconstitution of RNAi in S. cerevisiae
by introducing Dicer and Argonaute of Saccharomyces castellii
. They identified a novel class of Dicer protein present in S. castellii
which—unlike other known Dicer genes in Schizosaccharomyces pombe
, plants and animals—has two double-stranded RNA-binding domains (dsRBDs) but only a single RNaseIII domain and no helicase or PAZ domains. It has been proposed that S. castellii
Dicer may act as a homodimer and may not require additional dsRBDs. They also showed that the two genes Dicer and Argonaute of S. castellii
were sufficient to reconstitute the functional RNAi in S. cerevisiae
). Here we showed that the introduction of the two human genes Dicer and Ago2 did not enable RNAi in S. cerevisiae
without the concurrent presence of TRBP. The requirement for TRBP indicates a distinct domain and subunit architecture of RISC in human versus budding yeast. The reconstituted human RNAi system could facilitate functional dissection of human RNAi proteins and domains such as the helicase domain of Dicer and potential phosphorylation sites of TRBP.
In summary, we have shown that three human RNAi genes Ago2, Dicer and TRBP are sufficient to establish the human RNAi process in S. cerevisiae. The reconstituted human RNAi system in S. cerevisiae offers the potential for thorough molecular–genetic study of the regulation of human RNAi in an experimentally facile model eukaryote.