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. 2009 Oct;37(19):e127.
doi: 10.1093/nar/gkp657. Epub 2009 Aug 13.

RNA polymerase III can drive polycistronic expression of functional interfering RNAs designed to resemble microRNAs

Lindsey L Snyder  1 , Iqbal Ahmed, Laura F Steel
Affiliations

RNA polymerase III can drive polycistronic expression of functional interfering RNAs designed to resemble microRNAs

Lindsey L Snyder et al. Nucleic Acids Res. 2009 Oct.

Abstract

In both research and therapeutic applications of RNA interference, it is often advantageous to silence several targets simultaneously. Toward this end, several groups have developed vectors that utilize the model of endogenously encoded micro (mi) RNAs, where a single RNA polymerase II promoter can drive the expression of multiple interfering RNAs. Stronger pol III promoters have been used to drive individual short hairpin (sh) RNAs, but to date, it has been necessary to repeat the promoter in each silencing cassette to achieve multiplexed expression from a single vector. Here, we show that it is possible to drive polycistronic expression from a single pol III promoter when the interfering RNAs are formatted to resemble miRNAs rather than shRNAs. As many as four miRNAs designed to target hepatitis B virus (HBV) transcripts are shown to be processed and functional in reporter assays as well as in the context of replicating virus in cell culture systems. Although it has been observed that high levels of expression of shRNAs can lead to cytotoxicity, we find no significant evidence in transient transfection assays that the HBV-miRNAs produced by our vectors compete for the activity of endogenously produced miR-122 or for processing of an exogenously expressed miR-EGFP.

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Figures

Figure 1.
Figure 1.
Organization of cassettes for RNA pol III driven expression of miRNAs. (a) General structure of an expression cassette with a pol III promoter and restriction sites for insertion of DNA encoding a pre-miRNA. Facing block arrows (light gray) represent DNA encoding the stem portion and open bars show the flanking and loop sequences for each pre-miRNA. The shaded bar (dark gray) shows the pol III promoter and the (dT)6 pol III termination signal is indicated. The predicted structure of the pre-miRNA transcript is shown at the right, including sequence from the hsa-miR30 flanking regions that form a partially base paired structure at the base of the stem-loop necessary for recognition by the Drosha/DGCR8 microprocessor complex (40). Hash marks next to the stem-loop show predicted cleavage positions for processing to mature miRNA. (b) Structure of DNA encoding the bicistronic and polycistronic HBV-miRNAs and their RNA transcripts. DNA encoding the 1737B/1907A pre-HBV-miRNAs was inserted downstream of a pol III H1 or U6 promoter in a pUC-based plasmid to produce the bicistronic H1-2 and U6-2 silencing plasmids. Sequential insertion of XbaI/SpeI fragments encoding individual 2791B or 2910A pre-HBV-miRNAs produced the polycistronic cassettes H1-4 and U6-4. The position of insertion of individual HBV pre-miRNAs was changed to generate the rearranged cassettes, H1-4R and U6-4R. HBV-miRNAs are numbered according to their target site in the HBV genome. The expected structure of transcripts from each cassette is shown at the right, with individual HBV-miRNA stem-loops numbered (1–4) to highlight their order in the primary transcript.
Figure 2.
Figure 2.
Silencing activity of polycistronic miRNA constructs. (a) Huh7 cells were transfected with 250 ng of a dual luciferase reporter plasmid, psiCH-HBV, which contains sequence targeted by all four of the HBV-miRNAs, together with no silencing plasmid or increasing amounts of plasmids carrying the U6-2, U6-4, H1-2 or H1-4 HBV-miRNA expression cassettes, as indicated. Cotransfection with the reporter plasmid and 10 ng of a U6-miEGFP silencing plasmid (miEGFP) served as a negative control. Silencing activity was measured as the ratio of Renilla to firefly luciferase activity in cell lysates 2 days posttransfection, and results are reported as percent of control with no added silencing plasmid. Data represent the average (± SD) of triplicate assays of two separate transfections. (b) Huh7 cells were transfected with plasmids expressing two HBV-miRNAs from a pol II promoter [pCMV-30s-1737B/1907A and pLV-30s-1737B/1907A, see (15)] or the corresponding pol III driven cassettes (pUC-H1-2 and pUC-U6-2) and analyzed as in (a).
Figure 3.
Figure 3.
The silencing activity of individual HBV-miRNAs can be affected by their position in the polycistronic transcript. (a) Huh7 cells were transfected with 5 ng of monocistronic or polycistronic HBV-miRNA expression plasmids, together with 250 ng of a dual luciferase reporter plasmid carrying target for an individual HBV-miRNA, as indicated. (b) Huh7 cells were again cotransfected with monocistronic or polycistronic silencing plasmids together with a reporter plasmid carrying individual target sequences, as indicated. Here, the order of insertion of the HBV-miRNAs has been rearranged in the U6-4R construct relative to U6-4 (Figure 1). Data represent the average (± SD) of triplicate assays of two separate transfections.
Figure 4.
Figure 4.
Fully processed miRNA can be detected for each component of the polycistronic HBV-miRNA transcript. RNA was isolated from HEK-293T cells either not transfected or transfected with monocistronic or polycistronic vectors expressing HBV-miRNAs, as indicated above each lane. Two different blots (right and left panels) were probed sequentially for miRNAs or U6 RNA, as indicated to the right of each image.
Figure 5.
Figure 5.
Antiviral activity of the polycistronic HBV-miRNA plasmids. Huh7 cells were transfected with 100 ng pHBV/2, an infectious molecular clone of HBV, together with no silencing plasmid or increasing amounts of pUC-U6-4 or pUC-U6-4R, as indicated. pGLuc (50 ng) was included as a transfection efficiency control. Two days posttransfection, culture supernatants were assayed for HBV S-antigen (HBsAg) and results were normalized to secreted GLuc activity. Results are reported as percent of HBsAg secretion in the absence of silencing plasmid and represent the average (± SD) of two separate transfections assayed in triplicate.
Figure 6.
Figure 6.
The polycistronic HBV-miRNAs show minimal competition for activity of endogenous miR-122 in Huh7 cells or exogenously expressed miEGFP in HEK-293T cells. (a) Huh7 cells or HepG2 cells were transfected with psiCHECK-2 (psiCH2) or with psiCH2-122 where perfectly matched target sequence for miR-122 has been inserted downstream of the Renilla luciferase coding region. Results represent the average (± SD) of two separate transfections assayed in duplicate. (b) Huh7 cells were transfected with psiCH2-122 together with no silencing plasmid, or with increasing amounts of pUC-U6-2, pUC-U6-4 or pUC-H1-4 as indicated. As a control for reporter expression, cells were transfected with the psiCH2 plasmid, in the absence of silencing plasmid. Results show the average (± SD) of three separate transfections assayed in triplicate. For both (a) and (b), cells were lysed 2 days posttransfection and luciferase activity was assayed as in Figure 2. (c) HepG2 or Huh7 cells were transfected with psiCH-CAT360 or psiCH-CAT1454 together with the indicated amount of pUC-U6-2. Cells were lysed 3 days posttransfection and assayed as in Figure 2. Results show the average (± SD) of three separate transfections for HepG2 cells, and six transfections for Huh7 cells. (d) 293T cells were transfected with pCMV-dsEGFP in the absence of silencing plasmid, or with a constant amount of pUC-U6-miEGFP silencing plasmid plus increasing amounts of pUC-U6-2 or pUC-U6-4, as indicated. Immunoblot analysis of protein extracts prepared 2 days posttransfection was carried out using α-EGFP and α-actin antibodies. Duplicate lanes show results from two separate transfections for each set of experimental conditions. Numbers below each lane indicate the percent EGFP present relative to control (average of no miEGFP values) after normalization to actin.
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