To find the optimal construct for Pol II-directed synthesis of shRNA, we made a series of variants of human miR-30 hairpin structure (). We chose miR-30 as a model hairpin structure because its stem sequence could be substituted with other sequences that match different targets (20
). Because the previous studies suggested that simplifying miR-30 structure by eliminating the mismatch, the bulge and most of the base sequence does not significantly alter the RNAi efficiency (20
), we first constructed a similar hairpin and a series of its variations (, constructs 1–7). These hairpins targeted the human SOD1 mRNA and were placed downstream from the CMV promoter in an mRNA transcript. This construct design was similar to several previously published Pol II-shRNA synthesizing constructs (20
). In cotransfections with a report luciferase construct that contains the target sequence in its 3′-UTR, most of these hairpins (1–6 and 9) were expressed () and mediated RNAi against the target (), albeit all at lowered efficiency compared with the construct that carried the unmodified miR-30 hairpin structure (, construct 8; and ). The expression of construct 7 was not detected, nor was its RNAi efficacy ( and ). This lack of expression of shRNA in construct 7 is unlikely to be caused by RNA degradation as shown by ethidium bromide staining of this gel before transferring (data not shown). Rather, it might be caused by a lack of processing due to the relatively drastic change from the authentic miR-30 structure in this construct.
Figure 2 Comparisons in the levels of shRNA expression and in silencing efficacy. (A) Northern analysis of SOD1-shRNA expression under control of either Pol-II or Pol-III promoters. Each lane was loaded with equal amount of total RNA (20 μg/lane) extracted (more ...)
To determine whether RNAi efficiency might be further increased by including more sequences from the pri-miR-30 RNA that flank the hairpin, we tested a construct that contained 100 bp on either side of the miR-30 hairpin sequence from the pri-miR-30. In agreement with a previous report (30
), the RNAi efficiency was not further enhanced from hairpin 8 (data not shown), suggesting that the authentic miR-30 hairpin 8 contains the essential elements for RNAi mediated by Pol II-synthesized shRNA.
Thus, it is noticeable from the above results that those hairpins with fewer structural alterations from miR-30 have RNAi efficiency closer to the hairpin with the authentic miR-30 structure (, construct 8). For example, Hairpin 1 and 9, which had fewest structural alterations (), mediated RNAi slightly less efficiently than hairpin 8 (). In contrast, those with large structural alterations from miR-30 were far less effective or completely ineffective in RNAi (e.g. hairpins 6 and 7, see and ). Interestingly, the least effective shRNAs share the lacking of the distal A/C bulge from the stem ( and , hairpins 5–7), suggesting that this distal bulge is an important structural element of miR-30. Taken together, these results suggest that the authentic miR-30 structure is the most efficient in RNAi mediated by Pol II-synthesized shRNA in mammalian cells.
Our results differ from a previous study, in which a Pol II synthesized hairpin similar to hairpin 7 was shown to be processed correctly to form mature miRNA and mediate RNAi similar to hairpins with the authentic miR-30 structure (20
). The reason for this difference is not clear. One possibility is the difference in the mRNA sequences that harbor the hairpin between these two studies and these differences could lead to different processing of the transcripts. In terms of the RNAi efficiency, we note that in the previous experiments a high ratio of shRNA-synthesizing plasmid to the target plasmid (16:1 to 80:1) was used. Consequently, the RNAi effects might be saturated and the differences between the hairpins with the authentic miR-30 structure and other variations might not be revealed. In our experiments, the ratio of shRNA-synthesizing plasmid to the target plasmid was one to one.
Knowing that the authentic miR-30 hairpin structure was the best for the Pol II-directed hairpin expression and RNAi, we tested the efficacy of this hairpin by placing it in two additional constructs. In the first, we tried to enhance the RNAi efficacy by placing two hairpins targeting the same sequence in the human SOD1 gene downstream of the Pol II promoter (, construct 10) in order to produce more of the shRNA. This reasoning has precedents since some miRNA genes are located on the same chromosome as clusters with intervals as short as a few nucleotides and are expressed with similar profiles (18
). Such an organization suggests that these miRNA genes are expressed under control of the same promoter and their nascent transcripts are processed to become mature miRNAs. Recent experiments supported this notion (31
). Surprisingly, construct 10 failed to enhance the expression of shRNA and siRNA (), resulting instead in a decrease in RNAi efficacy ().
In the second construct, we placed the hairpin downstream of a different Pol II promoter, the human ubiquitin C promoter. This promoter has been used for constructing genes that were placed in transgenic mice and has been shown to efficiently express transgenes ubiquitously in multiple transgenic lines (25
). We placed the hairpin 8 in the first intron, which was part of the promoter cassette. Downstream of the first intron, we placed the eGFP open reading frame (, construct 11). The rationale for this design was based on recent evidence that some miRNAs are located in the non-coding regions or introns of mRNAs for known genes (18
). Since synthesis of both the shRNA and the eGFP depends on the same promoter, their expression in the same cells is assured. This provides a convenient way to monitor the cells in which the shRNAs are expressed, not only qualitatively, but also quantitatively. This UbC-shRNA-EGFP construct expressed shRNA at a similar level as the CMV promoter (construct 8) and mediated RNAi as efficiently (, construct 11; and ). Overall, the Pol III promoters still expressed at higher levels of shRNA and mediated stronger target silencing (, constructs 12 and 13; and ). However, the best Pol II constructs, 8 and 11, are approaching the levels of RNAi efficacy of the Pol III constructs ().
Can the Pol II-hairpin construct silence the endogenously expressed genes? To answer this question, we transfected the most effective hairpin constructs into the HEK293 cells. Compared with the controls, the best hairpin constructs 8 and 11 significantly inhibited the endogenous SOD1 expression (). Construct 11a, which was promoted by the UbC promoter but without the eGFP open reading frame, inhibited SOD1 expression similarly as construct 11, indicating that eGFP did not interfere with the silencing activity of construct 11. To further confirm this silencing, we tested the function of SOD1 in these cells. Since a lack of SOD1 is known to enhance cell's vulnerability to H2
), we treated the cells with H2
. Transfection with construct 8 and 11a significantly reduced the cell viability after the H2
treatment as compared with the control vectors (carrier vector and 8-mut) transfection ().
Figure 3 Pol II-directed synthesis of shRNA silenced the endogenous SOD1 expression and SOD1 function. (A) CMV and UbC promoter-directed synthesis of shRNAs knocked down the endogenous SOD1 in HEK 293 cells. HEK 293 cells were transfected with control and shRNA-expressing (more ...)
These experiments demonstrated that Pol II-directed synthesis of shRNA against SOD1 can mediate efficient RNAi. However, it was unclear whether this construct design was applicable to other target sequences and in different cell types. Furthermore, it remained unclear whether the eGFP expression provides a good indicator for the expression of the shRNA. To resolve these issues, we replaced the human SOD1 sequence in hairpin 8 with a sequence that target mouse Mn2+ superoxide dismutase (SOD2) () and placed these hairpin in construct 11 (). This construct along with the controls was transfected into NSC-34 cells. Because the transfection efficiency in these cells were lower (~50%) than in HEK293 cells (>90%), we selected clones that were transfected with the UbC-SOD2hp-eGFP vector. After expanding these clones, the SOD2 levels were measured. Different clones inhibited SOD2 expression differently (). Clone 3 inhibited SOD2 expression the most, followed by clones 5 and 4. The different levels of inhibition were correlated with the eGFP expression (, lower panels) and cellular toxicity. Clone 3 expressed the highest level of eGFP, followed by clones 5 and 4. Compared with the untransfected cells or cells transfected with the mutant SOD2 hairpin (clone 2), all cell clones that expressed SOD2 shRNA were unhealthy, the degree of which appeared more severe in clones 3 and 5 than clone 4 (, upper panels). Clones 3 and 5 expressed more eGFP than clone 4 (, lower panels). All clones that expressed eGFP and SOD2 shRNA could not be maintained for the long term, probably due to the cellular toxicity of SOD2 knockdown.
Figure 4 UbC-hp-EGFP provides a convenient indicator for shRNA expression. (A) The shRNA (antisense) sequence against Mn2+ superoxide dismutase (SOD2) was incorporated into the hairpin 8 structure and inserted into construct 11 (see ). A mutant hairpin (more ...)
To further confirm the quantitative correlation between the eGFP expression and the degree of silencing, we sorted cells that were transfected with construct 11 into four groups based on their eGFP expression levels. Proteins extracted from these cell groups were immunoblotted for eGFP and SOD2 (). The levels of eGFP were inversely correlated with the levels of SOD2, indicating that eGFP was a good indicator for the levels of shRNA expression and silencing efficacy.
Taken together, we have demonstrated that, by adopting the authentic miR-30 hairpin structure, Pol II can direct shRNA synthesis and mediate gene silencing with high efficiency comparable to Pol III. Therefore, this Pol II-hairpin design offers an alternative to Pol III constructs. Because the shRNA expression is under the control by same promoter as the indicator protein, the indicator protein indicates not only the cells that express the shRNA, but also the levels of the shRNA expression. This is especially valuable if an experiment desires to examine the effects of a gene expressed at different levels. Using construct 11, for example, one may separate cells with different levels of shRNA expression. Alternatively, one may establish the stably transfected cell lines that express different levels of shRNA using the sorted cells from the FACS, as demonstrated in . A further utility of this Pol II construct is that by replacing the ubiquitous Pol II promoter with various cell type-specific Pol II promoters, cell-specific gene silencing may be accomplished in vivo. In addition, various temporally inducible Pol II promoters, e.g. the tetracycline- or ecdysone-inducible promoters, can be used to temporally control gene silencing.