RESULTS
To assess the efficiency of siRNAs we designed a sensor that consisted of GFP fused with the viral target sequence for the siRNA. If the siRNA is active and efficient, the mRNA will be degraded leading to no or decreased GFP fluorescence signal compared to cells without siRNA expression.
Flow cytometry reveals suppression of EGFP expression by siRNA induction in yeast
Flow cytometry offers a means for efficient and precise evaluation of GFP expression at the individual cell level. In our experimental setup, we cultivated genetically modified yeast strains under tightly regulated environmental conditions. Subsequently, upon initiation of siRNA and GFP production, we proceeded to analyze each yeast cell culture using flow cytometry. This approach allows for precise measurement of any fluctuations in GFP signal. A decrease in GFP fluorescence indicates the effectiveness of the siRNA.
We used flow cytometry to measure the GFP fluorescence intensities in yeast cultures 24h after inducing the expression of shRNA and the GFP-target sequence reporter. In the absence of shRNA, the GFP-reporter-expressing cultures showed much higher GFP signal in comparison to background fluorescence of non-induced cultures, confirming sufficient expression of the reporter protein (Fig. 1). It is noteworthy that fluorescence intensities among different GFP-target constructs significantly varied, suggesting that the viral sequence introduced into the 3'-UTR of the transcript may influence mRNA stability (not shown). Reduced mRNA stability, in turn, leads to impaired translation and decreased GFP fluorescence. For this reason, to compare the impact of shRNAs on GFP reporter expression, we normalized the GFP fluorescence for each strain with shRNA to the data obtained for its parent strain without shRNA expression. Out of seven tested shRNAs, four showed statistically significant decrease in fluorescence levels, as determined by Two-sample Two-tailed Student’s T-test with unequal variances (p-value at least less than 0.05) (Fig. 1).
Figure 1. Expressing shRNAs in the engineered RNAi-capable yeast enables testing of siRNA activities. Plot showing the mean GFP fluorescence intensities of a population of cells expressing the indicated shRNA and the GFP reporters, measured by flow cytometry 24h after induction. The GFP fluorescence data presented was normalized to cells expressing the GFP reporter, but not shRNAs. The mean with standard deviation from 3 biological replicates for each shRNA variant is shown. Error bars indicate standard deviation. Statistical analysis (Two-sample Two-tailed Student’s T-test with unequal variances) was performed. Not Significant (NS) - p-value > 0.05; * - p-value ≤ 0.05; ** - p-value ≤ 0.01; *** - p-value ≤ 0.001.
These experiments lead to three important conclusions. Firstly, the presented work confirms the previously published results, demonstrating that introducing Dicer and Argonaute proteins to S. cerevisiae is sufficient to reconstitute RNAi response. Secondly, the experiments show that when viral sequences are introduced into the 3’-UTR of a GFP-coding transcript, they can be targeted by shRNAs in yeast, offering a straightforward means to assess the efficacy of various shRNAs. Thirdly, we observed partial suppression of GFP-reporter expression with tested anti-DWV siRNAs, making them strong candidates for further testing in bees to investigate possible off-target effects.
First round of experiments identifies points for further improvement in the yeast siRNA assay
Our results suggest that the viral target sequence can influence GFP reporter expression even in the absence of shRNAs. Although all the tested reporters have consistently demonstrated robust GFP expression, enabling the measurement of RNAi, it is advisable to explore further optimization of the viral sequence's length and its precise positioning for future investigations.
In our experimental setup, both shRNA and the GFP reporter are expressed from pGAL1 promoter, as was done in Drinnenberg et al 2009. However, it would be more advantageous to use different inducible promoters for shRNA and GFP. so that it would be possible to either express one or the other using the same strain. This would allow for the independent activation of either shRNA or GFP within the same strain. With the current single-promoter design, it's necessary to use different strains to observe reporter fluorescence without RNAi. By employing different promoters, the same strain with reporter expression activated but not shRNA could be utilized to measure reporter expression without inhibition. This modification would reduce the number of strains required for the experiment and simplify the methodology..
During the initial round of experiments, it was established that the tested shRNAs effectively induced RNA interference. However, it was not possible to detect quantitative differences in the efficiencies of these shRNAs, because most of them caused comparative partial suppression of the GFP reporter (Fig. 1). In these strains shRNAs are expressed from a high-copy-number 2μ plasmid, which drives extremely high expression. In future experiments, shRNAs could be expressed from a CEN plasmid that is present at a low number in yeast cells, resulting in lower expression of the shRNAs. This adjustment would enhance the method's sensitivity to detect and differentiate quantitative variations in shRNA efficiency.
