Engineering and optimization of the miR-106b cluster for ectopic expression of multiplexed anti-HIV RNAs

Abstract

Many microRNAs (miRNAs) are encoded within the introns of RNA Pol II transcripts, often as polycistronic precursors. Here, we demonstrate the optimization of an intron encoding three endogenous miRNAs for the ectopic expression of heterologous anti-HIV-1 small interfering RNAs (siRNAs) processed from a single RNA polymerase II primary miRNA. Our expression system, designated as MCM7, is engineered from the intron-embedded, tri-cistronic miR-106b cluster that endogenously expresses miR-106b, miR-93 and miR-25. Manipulation of the miR-106b cluster demonstrated a strict requirement for maintenance of the native flanking primary miRNA (pri-miRNA) sequences and key structural features of the native miRNAs for efficient siRNA processing. As a model for testing the efficacy of this approach, we have replaced the three endogenous miRNAs with siRNAs targeting the tat and rev transcripts of human immunodeficiency virus type 1 (HIV-1). This study has enabled us to establish guidelines for optimal processing of the engineered miRNA mimics into functional siRNAs. In addition, we demonstrate that the incorporation of a small nucleolar RNA TAR chimeric decoy (snoRNA) inserted within the MCM7 intron resulted in a substantial enhancement of HIV suppression in long-term acute infectious HIV-1 challenges.

FIG. 1. Overview of mir-106b polycistron based expression plasmids

(A) Schematic diagram of the mir-106b polycistronic miRNA expression plasmids and the derived siRNA expression plasmid used in this study. The “MCM7” name was used to indicate that the mir-106b cluster is located in intron 13 of the protein encoding gene MCM7 on chromosome 7q22.1. MCM7-E, -I and –S, refers to exon, intron and scaffold, respectively. MCM7-S1/S2/S3 refers to targeting of three independent targets sites, S1-S3, in the viral HIV-1 genome. U16TAR decoy refers to the nucleolar RNA TAR decoy, with forU16TAR being U16TAR expressed in the forward direction and revU16TAR in the opposite orientation. The constructs delSplice, miniS2 and miniMCM7, represent deletion variants of MCM7-S1/S2/S3. (B) Example of replacing the native miR stem-loop with an HIV-1 targeting shRNA sequence using the introduced restriction sites in MCM7-S, here illustrated with miR-106b and S1 using XhoI and HindIII. Mature miRNA/guide strand in green, predicted passenger strand (miR*) in red, heterologous sequences in uppercase, restriction sites in bold.

FIG. 1. Overview of mir-106b polycistron based expression plasmids

(A) Schematic diagram of the mir-106b polycistronic miRNA expression plasmids and the derived siRNA expression plasmid used in this study. The “MCM7” name was used to indicate that the mir-106b cluster is located in intron 13 of the protein encoding gene MCM7 on chromosome 7q22.1. MCM7-E, -I and –S, refers to exon, intron and scaffold, respectively. MCM7-S1/S2/S3 refers to targeting of three independent targets sites, S1-S3, in the viral HIV-1 genome. U16TAR decoy refers to the nucleolar RNA TAR decoy, with forU16TAR being U16TAR expressed in the forward direction and revU16TAR in the opposite orientation. The constructs delSplice, miniS2 and miniMCM7, represent deletion variants of MCM7-S1/S2/S3. (B) Example of replacing the native miR stem-loop with an HIV-1 targeting shRNA sequence using the introduced restriction sites in MCM7-S, here illustrated with miR-106b and S1 using XhoI and HindIII. Mature miRNA/guide strand in green, predicted passenger strand (miR*) in red, heterologous sequences in uppercase, restriction sites in bold.

FIG. 2. Functional expression of miRNAs from the MCM7 plasmid

(A) Knockdown of miR- and miR*-sensitive Renilla Luciferase reporters (psi-mir) after transient co-transfection of HCT116 cells with MCM7 plasmids. Rluc activity was normalized to the internal Firefly Luciferase control and values expressed relative to levels for control-transfected cells (± SD). (B) Northern analysis of endogenous expression levels of miR-106b, miR-93 and miR-25 in HCT116 cells (lane 1) and after transient transfection of MCM7-I (lane 2) and –E (lane 3). Lower and upper bands correspond to the mature miRNA and the pre-miRNA precursor, respectively. Arrow indicates mobility of a 20-nt RNA size marker. U6 snRNA was probed as an internal RNA loading control.

FIG. 3. Functional expression of siRNAs from the MCM7-S1/S2/S3 plasmid

(A) Knockdown of HIV-1 siRNA guide strand sensitive RLuc reporters (psi-Sx) after transient co-transfection of HEK293 cells with MCM7 plasmids. Rluc activity was normalized to the internal Firefly Luciferase control and values expressed relative to levels for control-transfected cells (± SD). (B) Northern analysis of siRNA guide strand expression levels after transient transfection of HCT116 cells with the MCM7-S1/S2/S3 construct. Arrow indicates mobility of a 20-nt RNA size marker. Note that for the Northern blot analysis, MCM7-S1/S2/S3 expression was driven from a Pol-II U1-promoter plasmid to avoid interference from the CMV-driven GFP marker which was used as a transfection control. U6 snRNA was probed as an internal RNA loading control.

FIG. 4. Limited efficacy of siRNAs expressed from MCM7-S1/S2/S3 deletion mutants

(A) Knockdown of siRNA-sensitive RLuc reporters after transient co-transfection of HEK293 cells with MCM7-S1/S2/S3 plasmids lacking the lower part of the miRNA stem-loop structure (miniSx). (B) Knockdown of siRNA-sensitive Renilla Luciferase reporters in MCM7- S1/S2/S3 plasmids lacking the flanking exon and the splice sites (delSplice) or missing the pri-miR-derived sequences between the stem-loop structures (miniMCM7). See Fig. 1A for construct diagrams. Rluc activity was normalized to the internal Firefly Luciferase control and values expressed relative to levels for control-transfected cells (± SD).

FIG. 5. Improved asymmetry of S2 siRNA RISC loading by a miR-93 structural mimic

(A) Knockdown of passenger strand-sensitive RLuc reporters (psi-S2-AS or psi-S2M-AS) after transient co-transfection of HEK293 cells with MCM7-S1/S2/S3 plasmid or a miR-93 mimic (S2M). Rluc activity was normalized to the internal Firefly Luciferase control and values expressed relative to levels for control-transfected cells (± SD). (B) Predicted stem-loop structure of miR-93, the fully base paired S2 hairpin and its miR-93 mimic, S2M. Mature miRNA/guide strand in green, predicted star/passenger strand in red, heterologous sequences in uppercase, restriction sites (EcoRI and BamHI) in bold.

FIG. 6. Optimized S3B containing cluster maintains the knockdown efficiencies of S1 and S2M, but has greatly improved S3 efficacy

Plasmids containing the three anti-HIV miRNA mimics were co-transfected with psi-CHECK vectors harboring the targets for each of the three siRNAs targeting S1, S2 and S3. The S3 unit was tested in the context of S1 and S2M in its original configuration (MCM7-S1/S2M/S3) or in the optimized configuration (MCM7-S1/S2M/S3B). The MCM7-S1/S2M/S3B cluster provides comparable levels of knockdown for sites S1 and S2, but demonstrated enhanced knockdown of the S3 target (A). Good strand selectivity is maintained for MCM7-S1/S2M/S3B when the target is inserted in the anti-sense orientation (B). The data are presented as the means of triplicate measurements. (C) Northern analysis of siRNA guide strand expression levels after transient transfection of HCT116 cells. RNA from MCM7-S1/S2/S3 (lane 1) and MCM7-S1/S2M/S3B (lane 2) transfected cells, respectively. Arrow indicates mobility of a 20-nt RNA size marker. U6 snRNA was probed as an internal RNA loading control.

FIG. 6. Optimized S3B containing cluster maintains the knockdown efficiencies of S1 and S2M, but has greatly improved S3 efficacy

Plasmids containing the three anti-HIV miRNA mimics were co-transfected with psi-CHECK vectors harboring the targets for each of the three siRNAs targeting S1, S2 and S3. The S3 unit was tested in the context of S1 and S2M in its original configuration (MCM7-S1/S2M/S3) or in the optimized configuration (MCM7-S1/S2M/S3B). The MCM7-S1/S2M/S3B cluster provides comparable levels of knockdown for sites S1 and S2, but demonstrated enhanced knockdown of the S3 target (A). Good strand selectivity is maintained for MCM7-S1/S2M/S3B when the target is inserted in the anti-sense orientation (B). The data are presented as the means of triplicate measurements. (C) Northern analysis of siRNA guide strand expression levels after transient transfection of HCT116 cells. RNA from MCM7-S1/S2/S3 (lane 1) and MCM7-S1/S2M/S3B (lane 2) transfected cells, respectively. Arrow indicates mobility of a 20-nt RNA size marker. U6 snRNA was probed as an internal RNA loading control.

FIG. 7. Expression of the RNAs in the optimized MCM7 constructs

HEK293 cells were transiently transfected with each of the optimized MCM7 constructs and controls. Forty-eight hours following transfection, total RNA was collected, electrophoresed in an 8% polyacrylamide gel with 8M urea, blotted onto a nylon membrane, and hybridized with the corresponding ³²P-labled probes. Hybridization to U6-driven short hairpin siRNAs S1, S2, S3, and the U16TAR decoy expressed from Shrek 3 were used as positive controls. RNA prepared from an empty vector (pcDNA) transfected cell line was used as a negative control. S1, S2, and S3 siRNAs are approximately 21 nucleotides. The U16TAR decoy is 132 nucleotides.

FIG. 8. Anti-HIV-1 activity of the optimized MCM7 constructs

HEK293 cells were co-transfected with the indicated vectors and HIV-1 NL4-3 at a ratio of 4:1. The empty pcDNA vector served as the negative control. To demonstrate a higher combinatorial knockdown efficacy, the MCM7 constructs were compared to individually expressed Pol III hairpins: shS1, shS2, and shS3. Culture supernatants were collected at 24, 48, and 72 hour time points, analyzed by the bDNA assay (Panomics), and values are depicted as means ± standard deviation. As compared to the MCM7-S1/S2M/S3B construct, the addition of the TAR decoy in both the reverse and forward orientations provided a two fold enhancement in the inhibition of HIV-1 replication and was also more potent than the individual Pol III shRNA expression units.

FIG. 9. Addition of the U16TAR decoy confers greater anti-HIV-1 activity

Stably transduced CEM-T cells (A) and SupT1 cells (B) expressing the indicated vectors were challenged with HIV-IIIB and HIV-LAI, respectively. Culture supernatants were collected at various time points and analyzed by a p24 ELISA assay. Data points are reported as means ± standard deviation. Standard deviation calculations for each data point in panel (A) were <1.3. The addition of the U16TAR decoy in the MCM7-S1/S2M/S3B revU16TAR (closed square) confers long-term viral inhibition as compared to the MCM7-S1/S2M/S3B construct (closed circle).